WO1999009150A1

WO1999009150A1 - Method of introducing modifications into a gene

Info

Publication number: WO1999009150A1
Application number: PCT/US1997/014507
Authority: WO
Inventors: Dana Owen Wirak
Original assignee: Bayer Corporation
Priority date: 1996-08-15
Filing date: 1997-08-19
Publication date: 1999-02-25

Abstract

The present invention relates to a gene targeting vector and a method of using it to modify nucleic acid sequences. A gene targeting vector in accordance with the invention can comprise: a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a target gene, operably linked to the 5' terminus of a nucleotide coding sequence which, when inserted into a target gene, codes for at least one amino acid whose identity and/or position is not naturally-occurring in the target gene, and a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of the target gene, operably linked to the 3' terminus of said nucleotide coding sequence. The nucleotide coding sequence can code without interruption for an amino acid sequence, where the amino acid sequence is coded for by two or more exons in a naturally-occurring gene.

Description

METHOD OF INTRODUCING MODIFICATIONS INTO A GENE

BACKGROUND

The current prior art methods of modifying genes are cumbersome and difficult. For example, mutagenesis of the mouse gene locus via "hit-and- run" and "tag-and-exchange" gene targeting technologies can require the mouse gene locus to be targeted two times in succession using the same ES cell clone. This is a long and laborious process. It is extremely difficult to maintain totipotency of the ES cell through so many manipulations and over such long periods of time in culture. To overcome the difficulties in the prior art, we have developed a novel method of targeting and engineering gene sequences.

The current state of the art provides for three different approaches to the development of transgenic animal models (Lamb, Nat. Genet. , 9:4-6, 1995). The first approach utilizes pronuclear injections of recombinant minigenes into the pronuclei of 1-cell embryos. In the second approach, a complete gene residing in yeast artificial chromosomes (YACs), is electroporated into embryonic stem cells (ES cells). The third approach utilizes gene targeting techniques in ES cells to introduce point mutations into a gene present in the ES cell chromosome. The most common approaches to introducing point mutations are "hit-and-run" (Hasty et al., Nature, 350:243-

246, 1991) or "tag-and-exchange" (Askew et al., Mol. Cell. Biol. , 13:4115- 4124, 1993), (Stacey et al., Mol. Cell. Biol , 14: 1009-1016, 1994) gene targeting procedures.

Recombinant minigenes, when injected into mouse embryos, integrate into the mouse chromosome at random locations. The site of integration can often exert a deleterious influence on the pattern of expression and/or expression level of the recombinant level of the recombinant minigene ("position effect") (Bonnerot et al., Proc. Natl. Acad. Sci. , 87:6331-6315, 1990; Brinster et al., Proc. Natl. Acad. Sci. , 85:836-840, 1988; Grosveld et al., Cell, 51:976-985, 1987).

To illustrate the benefit and ease of the novel compositions, methods, treatments, etc., described herein, we have utilized genes associated with Alzheimer's disease. Therefore, although aspects of this disclosure are written with respect to Alzheimer's diseases, e.g., the APP gene, it is recognized that this invention is in no way limited to such genes and diseases, but may be applied to any nucleic acids., etc., that one desires to target and/or modify. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of memory and cognition. Prominent his- topathological features of this disease include the extracellular deposition of amyloid and the accumulation of intracellular neurofibrillary tangles. The principal underlying cellular features of AD are the degeneration affects many types of neurons and may account for the numerous neurological deficits that patients afflicted with the disease encounter. The most notable degeneration occurs in the hippocampus, cerebral cortex, and amygdala, regions of the brain that play a major role in memory, cognition, and behavior.

Although numerous attempts have been made to generate transgenic mouse models for AD via the pronuclear injection approach (Lamb, 1995), only one line of transgenic mice has succeeded in developing extra-cellular plaque-like deposits of beta-amyloid (Games et al., Nature, 373:523-527, 1995). This transgenic mouse line utilizes the PDGF promoter to over- express (> 10 fold) human "London" -FAD APP. Because of the artificial nature of the transgene's regulation of gene expression and the aberrant high levels of APP expression, the accumulation of amyloid in this line of transgenic mice may not be fully relevant to the cellular mechanisms involved in Alzheimer's disease. Two additional papers report only partial success in developing AD- like pathology. In one transgenic model, human APP 751 is over-produced in the brain using the brain-specific enolase promoter (Higgins et al., Ann. Neurol. , 35:598-607, 1994). This mouse model exhibits diffuse extra-cellular staining for beta-amyloid, but there was no evidence of accumulations of plaque-like deposits as described by Games et al. (Games et al., 1995). Another transgenic model exhibits intra-cellular deposits of beta-amyloid (La Ferla et al., Nat. Genet. , 9:21-30, 1995). This deposition leads to neuropathological processes, including apoptotic neurons and gliosis. All transgenic mice derived via pronuclear injections retain the ability to express mouse APP. It has been demonstrated that mouse amyloid peptides do not aggregate in solution nearly as well as the human amyloid peptides (Dyrks et al., FEBS Lett. , 324:231-36, 1993). It is likely that the mouse amyloid peptide interferes with the process of human amyloid aggregation. This may, in part, explain the necessity in the existing mouse

AD model to greatly over-express human amyloid in a mouse brain to develop extra-cellular amyloid deposits.

The human APP gene locus encompasses a very large region (~400 Kb). Transgenic mice have been generated using YACs which appear to contain an intact human APP gene (Lamb et al., 1993; Pearson and Choi,

Proc. Natl. Acad. Sci., 30:10578-10582, 1993). But because gene regulatory elements have been identified at considerable distances from the proximal promoter of many genes (e.g., (Grosveld et al., 1987) and (Simonet et al., J. Biol. Chem. , 268:8221-8229, 1993)) there is no assurance that a given APP YAC clone contains all necessary APP gene regulatory elements. AD is a complex disease of aging, and the regulation of APP gene expression may play a critical role in the onset and progression of the disease. An accurate mouse model for AD may very well require the presence of critical APP gene regulatory elements which may be missing or altered in the YAC clones. In addition, the YACs will integrate at random sites in the mouse chromosome after electroporation and expression of the human APP gene may be altered in a detrimental fashion due to "position" effects (see above).

YAC clones are inherently unstable and it can be very difficult to generate transgenic mouse lines where the gene locus resident on the YAC has remained intact. Furthermore, FAD mutations need to be introduced into the very large YACs via homologous recombination in yeast. Determining the stability and integrity of FAD- APP YACs require considerable effort (Lamb et al., 1993, Pearson and Choi, 1993).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of modifying a target nucleic acid. The target nucleic acid preferably comprises a genomic DNA sequence. The invention also relates to recombinant nucleic acid molecules which comprise a nucleotide sequence effective for homologous recombination at a predefined position of a gene and which is operably linked to a nucleotide coding sequence. Such recombinant nucleic acid molecules can be further combined with a vector sequence, a selectable marker, etc., to form a targeting vector useful for modifying a target nucleic acid, e.g., a genomic DNA sequence. The invention also relates to transgenic animals which comprise cells containing a recombinant gene, e.g., an APP gene or a presenilin gene, where the gene has been modified or engineered using the mentioned gene targeting vector. The transgenic animals are useful as animal models for diseases associated with the modified gene locus, e.g., Alzheimer's disease for the APP or presenilin genes. An object of the invention is a novel gene targeting strategy that facilitates the introduction of one or more specific mutations into any gene in a single double reciprocal homologous recombination step, providing a clear advantage over other gene targeting approaches which use at least two transfection and screening/selection steps. The gene targeting strategy preferably utilizes double reciprocal homologous recombination and a positive selectable marker gene to facilitate the insertion of gene segments or cDNA's (from the same or a heterologous host) into specific sites within the chromosome of a desired host cell, e.g., an embryonic stem (ES) cell derived from a rodent such as mouse. By the term "cDNA", it is meant a DNA which has been obtained by copying mRNA. The gene segments or cDNA's can be modified to encode one or more mutations. These gene-to-gene segments or gene-to-cDNA fusions, therefore, allow the introduction of one or more specific mutations into the coding sequence of the targeted gene. For some purposes, it may be preferable to employ a cDNA which is modified by the addition of other desired sequences, either coding or non-coding.

An aspect of the invention is a recombinant nucleic acid molecule comprising a nucleotide coding sequence, e.g., a cDNA, which is operably linked at its 5' or 3' terminus, or at both, to a nucleotide sequence which is effective to achieve homologous recombination. The invention also relates to a nucleotide sequence of a rodent APP gene such as a murine APP gene, or other mammal, which is effective to achieve homologous recombination at a predefined position in a target gene, operably linked to the 5' terminus, 3' terminus, or both, of a nucleotide sequence coding for at least one amino acid which is not naturally occurring at a specific amino acid position of the target gene. When the molecule comprises sequences at its 5' and 3' terminus which are homologous to the target gene, the molecule is effective to achieve homologous recombination with the target gene located, e.g., on a chromosome.

The term recombinant means a nucleic acid molecule which has been modified by the hand-of-man, e.g., comprising fragments of nucleic acid from different sources or a nucleic acid molecule from one source which has been engineered. Thus, the nucleic acid molecule is recombinant, e.g., because it comprises nucleotide sequences from a rodent (e.g., mouse) gene and a human gene or a synthetic (i.e., engineered) nucleotide sequence. Homologous recombination is a process in which nucleic acid molecules with similar genetic information line up side by side and exchange nucleotide strands. A nucleotide sequence of the recombinant nucleic acid which is effective to achieve homologous recombination at a predefined position of a target gene therefore indicates a nucleotide sequence which facilitates the exchange of nucleotide strands between the recombinant nucleic acid molecule at a defined position of a target gene, e.g., a mouse APP gene. The effective nucleotide sequence generally comprises a nucleotide sequence which is complementary to a desired target nucleic acid molecule (e.g., the gene locus to be modified), promoting nucleotide base pairing. Any nucleotide sequence can be employed as long as it facilitates homologous recombination at a specific and selected position along the target nucleic acid molecule. Generally, there is an exponential dependence of targeting efficiency on the extent or length of homology between the targeting vector and the target locus. Selection and use of sequences effective for homologous recombination is described, e.g., in Deng and Capecchi, Mol. Cell. Bio. , 12:3365-3371, 1992; Bollag et al., Annu. Rev. Genet. , 23:199-225, 1989; Waldman and Liskay, Mol. Cell. Bio., 8:5350-5357, 1988.

The nucleotide sequence effective for homologous recombination can be operably linked to a nucleotide sequence, preferably comprising a nucleotide coding sequence, which is to be recombined into the desired target nucleic acid. For example, an aspect of the present invention is to replace all or part of the amino acids comprising exons 16, 17, and 18 of the APP gene with a cDNA coding for all or part of the corresponding amino acids of a human APP gene. This is achieved by attaching a part of the APP gene comprising a part of intron 15 and exon 16 to the 5' terminus of a human cDNA and a part of the APP gene comprising a part of intron 16 to the 3' terminus of the cDNA to form a targeting vector. The APP gene segments are positioned with respect to the human cDNA in a way such that homologous recombination between them and the mouse gene will result in replacement of exons 16 through 18 with the cDNA. Such positioning, i.e., operable linkage, means that the mouse gene segment is joined to the cDNA whereby the homologous recombination function can be accomplished. A nucleic acid comprising a nucleotide sequence coding without interruption means that the nucleotide sequence contains an amino acid coding sequence for a polypeptide, with no non-coding nucleotides interrupting or intervening in the coding sequence, e.g., absent intron(s) or the noncoding sequence, as in a cDNA.

An object of the present invention is to introduce modifications into genomic sequences, e.g., by introducing into or replacing a genomic sequence with a cDNA. Such cDNA can comprise one or more mutations, thereby facilitating the introduction of any desired nucleotide sequence into a target nucleic acid. The introduced nucleic acid, e.g., a DNA can particularly encode modifications in, or which span, two or more exons of a desired gene using only a single, double reciprocal homologous recombination event. In one embodiment, two independent point mutations can be introduced into a genomic sequence, where each point mutation is located in a different exon of the same gene. Thus, the coding sequence can be a nucleotide sequence which codes without interruption for an amino acid sequence, where the amino acid sequence is coded for by two or more exons in a naturally-occurring genomic (i.e., gene) sequence. This includes, e.g., a coding sequence for an amino acid sequence which is a cDNA, where the cDNA comprises amino acids coded for by separate exons of a naturally- occurring genomic sequence comprising exons and introns. By the phrase naturally-occurring genomic sequence, it is meant the gene structure as it occurs in nature. For example, a human APP gene contains 18 exons in a naturally-occurring form which has been described. See, e.g., Yoshikai et al., Gene, 87:291-292, 1990. Other gene structures are also possible.

The introduction of point mutations via a replacement type vector has been described (Rubinstein et al., Nucl. Acid Res. , 21 :2613-2617, 1993). Rubinstein et al. did not consider fusing genomic sequences with cDNA sequences to encode the gene product. Therefore, the gene targeting technology described by Rubinstein et al. would not succeed in introducing the Swedish-London and Swedish-714 stop double mutations into the mouse APP gene locus. The beta-amyloid domain resides on two separate exons (Lemaire et al. , Nucleic Acid Res. , 17:517-522, 1989; Kang and Muller-Hill,

Biochem. Biophys. Res. Comm. , 166:1192-2000, 1990). While the Swedish mutation and human amino acid differences reside on exon 16, the London mutation resides on exon 17. Lambda genomic clones are not large enough to encompass both exons (Lamb et al., Nature Genetics, 5:22-30, 1993). Therefore, the introduction of the double mutations into a host gene locus

(e.g., a mouse APP gene) by the previously described gene-targeting approaches would require multiple gene targeting events utilizing two independent targeting vectors. Thus, it is recognized that in accordance with the present invention mutations which span sequences too large to fit into conventional vectors, targeting strategies, etc. (such as described in Lamb et al., 1993), e.g., two or more exons, can be introduced into genomic DNA by preparing targeting vectors comprising an intron effective for homologous recombination and a contiguous coding sequence, e.g., from the two or more exons. The nucleotide coding sequence can code for at least one amino acid whose identity and/or position is not naturally-occurring in a target gene, e.g., a rodent (e.g., mouse) or non-human mammal gene. This means that the nucleotide coding sequence, when inserted into the target gene such that an open reading frame is formed with the target gene coding sequences, contains at least one non-identical amino acid from the coding sequence of the unmodified target gene. This can mean amino acid substitution, deletion, or addition. In the examples below which illustrate, but in no way limit the invention, a nucleic acid coding for amino acids of a mouse APP gene are replaced by nucleic acid coding for amino acids of a human APP gene. At least 5 alternative splice forms of APP have been detected (reviewed in Beyreuther et al., Ann. NY Acad. Sci., 695, 91-102 (1993)). The amino acids of a human APP gene means amino acid(s) identified as non-identical when the two APP gene sequences are compared. The amino acid numbering in the patent application refers to the largest alternative splice form of APP which consists of 770 amino acids. See, e.g., Kitaguchi et al., Nature 331, 530-532 (1988); Tanaka et al., Biochem. Biophys. Res. Commun., 157, 472-479 (1988). For example, the human amino acid sequence differs in the beta-amyloid domain are at positions 676, 681, and 684. The mouse APP gene contains a gly cine at amino acid position 676, and a phenylalanine at amino acid position 681, and an arginine at amino acid position 684. A nucleotide coding sequence, which when inserted into an open reading frame of the mouse APP gene, comprising an arginine at amino acid position 676, a threonine at amino acid position 681, and/or a histidine at amino acid 684 is considered to contain three amino acid(s) whose identify is not naturally- occurring at an amino acid position (i.e., 676, 681, and/or 684) in the target mouse APP gene. See Figure 17 for other differences between the mouse and human APP polypeptide sequence.

A nucleic acid coding for at least one amino acid not naturally occurring in the targeted gene can also comprise, e.g., nucleotides which occur in a naturally-occurring human gene, such as naturally-occurring polymorphisms, alleles, or mutations which are discovered or identified in a natural population. By the term naturally-occurring, it is meant that the nucleic acid is obtained from a natural source, e.g., animal tissue and cells, body fluids, tissue culture cells, forensic samples. Any other amino acid(s) can be incorporated, as well as, e.g., conservative and non-conservative amino acid substitutions, amino acid(s) obtained from other genes, non- naturally-occurring or engineered sequences, functional and/or selectable coding sequence domains.

In the examples, a mouse APP gene is targeted by the substitution of an amino acid found in a human APP gene. Numerous naturally-occurring mutations have been identified in non-murine APP genes. A nucleic acid according to the present invention can contain such mutations. Other modifications to the sequence can comprise mutations found in familial or genetic cases of disease, preferably Alzheimer's disease, Down's syndrome, or heredity cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). A nucleotide sequence coding for all or part of an amino acid sequence of a human APP gene can contain codons found in a naturally-occurring gene or transcript, or it can contain degenerate codons coding for the same amino acid sequences.

Preferred human APP amino acid sequences include: Swedish-FAD, KM(670,671)NL; London-FAD, V(717)I; Swedish/London-FAD, KM(670,671)NL, V(717)I; stop codon at position 714; Swedish-FAD, KM(670,671)NL, stop codon at position 714, etc. See Table 1.

An amino acid sequence of a human APP gene comprising a nucleotide sequence to be inserted into a targeted mouse APP gene preferably codes without interruption and comprises arginine at 676, threonine at position 681, histidine at position 684, or combinations thereof, in addition to other mutations and engineered codons.

The present invention also relates to nucleic acids which hybridize to a nucleic acid coding for an amino acid sequence of a human APP gene, preferably under stringent conditions. Such hybridizable sequences are preferably not a naturally-occurring mouse APP nucleotide sequence; however, mutant mouse APP sequences can be included.

Hybridization conditions can be chosen to select nucleic acids which have a desired amount of nucleotide complementarity with the nucleotide sequence coding for all or part of an amino acid sequence of a human APP gene. A nucleic acid capable of hybridizing to such sequence, preferably, possesses 50%, more preferably, 70% complementarity, between the sequences. The present invention particularly relates to nucleotide sequences which hybridize to the nucleotide sequence coding for human APP amino acids under stringent conditions. As used here, "stringent conditions" means any conditions in which hybridization will occur where there is at least about 95%, preferably 97%, nucleotide complementarity between the nucleic acids. A nucleotide sequence hybridizing to the coding sequence will have a complementary nucleic acid strand, or act as a template for one in the presence of a polymerase (i.e. , an appropriate nucleic acid synthesizing enzyme), which has a corresponding amount of nucleotide identity or similarity. The present invention includes both strands of nucleic acid, e.g., a sense strand and an anti-sense strand. Thus, it is understood that a nucleic acid comprising a nucleotide sequence hybridizing to the coding nucleotide sequence of amino acids of a human APP gene also represents a nucleic acid which possesses at least about 95%, preferably 97% nucleotide sequence identity.

According to the present invention, at least one amino acid not naturally-occurring in the targeted gene also includes amino acids selected from engineered or non-naturally-occurring sequences. In the examples, a mouse APP gene is modified by replacing mouse amino acids with amino acids which naturally occur in a human APP gene. However, the mouse APP gene can also be modified or engineered by the introduction of amino acids which are not based on a human APP gene, e.g., conservative or non- conservative amino acids, cysteines, prolines, functional and/or selectable domains, etc.

Changes or modifications to the nucleotide coding sequence can be accomplished by any method available, including directed or random mutagenesis to a nucleic acid. These sequence modifications include, e.g., nucleotide substitution which does not affect the amino acid sequence (e.g., different codons for the same amino acid), replacing naturally-occurring amino acids with homologous or conservative amino acids, e.g. (based on the size of the side chain and degree of polarization), small nonpolar: cysteine, proline, alanine, threonine; small polar: serine, glycine, aspartate, asparagine; large polar: glutamate, glutamine, lysine, arginine; intermediate polarity: tyrosine, histidine, tryptophan; large nonpolar: phenylalanine, methionine, leucine, isoleucine, valine. In addition, it may be desired to change the codons in the sequence to optimize the sequence for expression in a desired host.

In addition to a gene segment effective for homologous recombination and coding sequence to be recombined, e.g., a recombinant nucleic acid molecule according to the present invention also can include selection markers, 3' regulatory sequences, regulatory sequences, restriction sites, vector sequences, and sequences and/or modification which enhance homologous recombination.

In order to identify cells which have integrated the nucleic acid molecule, it is desirable to include a selectable marker gene, e.g., neomycin resistance, gene HPRT gene, etc. A selectable marker gene codes for a product which can be directly or indirectly detected in a host in which it is expressed. Selectable marker genes and their use are widely used in molecular biology. When a neomycin resistance gene is utilized, cells having incorporated it can be selected by resistance to G418. A second selectable marker gene can also be incorporated into the vector, e.g., a herpes simplex virus thymidine kinase gene. Any selectable genes routinely used in host cells can be used in the gene targeting vectors, including HSV TK, neo^r, hygromycin, histidinol, Zeocin (Invitrogen), HPRT, etc. Selectable genes can also be included to select against random integration events. Thus, selection for the first marker (e.g., by positive selection), and absence of the second marker (e.g., by negative selection), permits enrichment for transformed cells containing a modified target nucleic acid sequence, e.g., at the APP gene locus. The choice and arrangement of the selectable marker gene(s) in the recombinant nucleic acid molecule are as the skilled worker would know, e.g., described in U.S. Pat. No. 5,464,764 and Rubinstein et al., Nucl. Acid Res. , 21:2613-2617, 1993. A preferred recombinant nucleic acid comprises a selectable marker gene, e.g., a gene for neomycin resistance, in the mouse APP gene segment 3' to the cDNA. The selectable marker genes can be operably linked to regulatory sequences which control their expression, e.g., in a cell or tissue specific manner. Examples of such sequences are described, e.g., in U.S. Pat. No. 5,464,764.

In accordance with the present invention, 3 ' regulatory nucleotide sequences can be operably linked to a recombinant nucleic acid molecule. For example, it may be desirable to include a transcription termination signal and/or polyadenylation signal (e.g., AATAAA tandem repeat) at the 3' end of the nucleotide sequence to be inserted into the foreign gene. Generally, a selectable marker gene directly follows the transcription, termination and polyadenylations signals. Other sequences can also be included, e.g., nucleotide sequences which regulate the stability of a mRNA. A recombinant nucleic acid can also comprise nucleotide sequences which affect expression of the gene into which it is combined, e.g., enhancers.

A recombinant nucleic acid molecule according to the present invention can also comprise all or part of a vector. A vector is a nucleic acid molecule which can replicate autonomously in a host cell, e.g., containing an origin of replication. Vectors can be useful to perform manipulations, to propagate, and/or obtain large quantities of the recombinant molecule in a desired host. A skilled worker can select a vector depending on the purpose desired, e.g., to propagate the recombinant molecule in bacteria, yeast, insect, or mammalian cells. Examples of useful vectors include Bluescript KS+II (Stratagene). The following vectors are provided by way of example, Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs., pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).

Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other vector, e.g., plasmids, viruses, or parts thereof, may be used as long as they are replicable and viable in the desired host. The vector can also comprise sequences which enable it to replicate in the host whose genome is to be modified. The use of such vector can expand the interaction period during which recombination can occur, increasing the targeting efficiency.

Recombinant nucleic acid molecules according to the present invention can also include sequences and modifications which decrease nonhomologous recombination events and/or enhance homologous recombination. For example, it has been found by Chang & Wislon, Proc. Natl. Acad. Sci. USA, 84:4959-63, 1987, that the addition of dideoxy nucleotides to the recessed termini of DNA molecules could enhance homologous recombination 6- to 7- fold relative to nonhomologous events. Recombinant nucleic acid molecules according to the present invention can be prepared according to the various methods known to the skilled worker in the art, e.g., as mentioned in Current Protocols in Molecular Biology, Edited by F.M. Ausubel et al., John Wiley & Sons, Inc; and Current Protocols in Human Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc.

In accordance with the present invention, the novel gene-targeting can be used to modify any desired gene. Figure 15 illustrates several general strategies. Figure 15 A shows a "typical" host gene with a DNA sequence consisting of a gene promoter, a series of exons (5 in this example). The exons are depicted as boxes. The gene can contain one or more exons. The line between the boxes (exons) represent the introns. The 5' -end of each intron contains a splice donor site which lies directly juxtaposed to the 3'- nucleotide to the preceding exon. The 3 '-end of each intron contains a splice acceptor sequence which lies directly juxtaposed to the 5' -end of the neighboring exon. The 3' -end of the last exon contains a nonsense codon (designated as a stop) to terminate translation. This is followed by 3'- untranslated sequences which are present in the gene transcript and then a transcription termination and polyadenylation signal (designated poly A).

Figure 15B illustrates a targeted gene where a cDNA is inserted directly into an exon (exon 4 in this example) of the gene. Using an appropriately designed gene-targeting construct, any exon of a mouse gene can be targeted in this fashion. This is the approach used in the examples to generate the Swedish and/or London FAD-m/hAPP transgenic mice. The sequence of the cDNA is arranged so that the fusion between the gene and the cDNA creates an "in-frame" sequence that properly encodes the desired protein. The cDNA can be modified to encode one or more mutations (designated at ^*). The cDNA can be derived from transcripts from other genes of the same species or from genes from other species.

The cDNA is inserted into the mouse by homologous recombination. The recombination occurs between the targeted gene and an exogenously added gene-targeting construct or vector. The vector is preferably linearized. For homologous recombination to insert the cDNA into the proper location and orientation within the targeted gene, the DNA components of the vector can be arranged in a specific manner. The cDNA is preferably positioned between nucleotide sequences which are homologous to specific locations of the targeted gene. In the gene-targeting vector, there is preferably a gene segment comprising a nucleotide sequence corresponds substantially to an upstream (5 '-flanking) region of the targeted gene. This segment comprises contiguous and sufficient upstream (5 '-flanking) sequences of the targeted gene to allow efficient recombination to take place, i.e., a nucleotide sequence which is effective for homologous recombination. The segment can be followed by a portion of the targeted gene exon (exon 4 in this example).

In the gene-targeting vector, the sequence spanning the junction between the 3 '-end of the targeted gene exon (exon 4 in this example) and the 5-end of the cDNA are arranged precisely in-frame to conserve the open reading frame to properly encode the desired gene product. In effect, the cDNA and the exon into which it is inserted become the terminal exon of the targeted gene. For proper termination and maturation of the transcript encoded by the targeted gene, transcription termination and polyadenylation signals (designated poly A) are positioned directly after the cDNA (and after the translation of stop codon). Directly following the transcription termination and polyadenylation signals, the gene targeting vector further comprises a selectable marker gene such as the neomycin resistance gene (designated neo¹) or the HPRT gene.

The gene targeting vector can further comprise a downstream (3'- flanking) region of homology to the targeted gene which is placed directly after the selectable marker gene, e.g., neo^r. The downstream region of homology can comprise contiguous gene sequences but can be any length of sequence providing it is sufficiently long to facilitate homologous recombination. The 5 '-end of the downstream region of gene homology can be located at any position proximal to the targeted gene as long as it lies downstream (3') of the mouse gene sequence which forms the junction between the targeted gene exon and the cDNA. After homologous recombination has taken place, the DNA sequence of the targeted gene positioned between the 3 '-end of the upstream region of gene homology and the 5 '-end of the downstream region of gene homology will have been deleted.

After homologous recombination takes place, exon sequences lying 5 ' of the exon/cDNA junction will encode the N-terminal portion of the gene product while the cDNA sequences lying 3' of the exon/cDNA junction will encode the C-terminal portion of the gene product.

Figure 15C illustrates a targeted gene where a cDNA is inserted directly into an intron (intron 3 in this example) of the targeted gene. Using an appropriately gene-targeting construct, any intron of a gene could be targeted in this fashion.

The sequence of the cDNA is arranged so that it functions as the terminal exon of the targeted gene. To form an open reading frame between the targeted and human coding sequence, the codon reading-frame of the cDNA sequence is positioned in-frame with the codon reading-frame of the nearest upstream (5') exon (exon 3 in this example). For proper splicing of messenger RNA to occur, a functional splice acceptor site immediately preceding (5') the cDNA can be included. Thus, after splicing of the exon with the cDNA, a resultant transcript from the targeted gene will encode the desired gene product. As mentioned above, a cDNA from various sources can be utilized and it can be modified to encode mutations. The arrangement of the gene targeting vector is as described above.

Figure 15D illustrates a targeted gene where a gene segment from another the same or different species (designated as foreign gene segment) is inserted directly into an intron (intron 3 in this example) of the targeted gene. Using an appropriately gene-targeting construct, any intron of a gene can be targeted in this fashion. In this example, the sequence of the foreign gene segment contains normal exons and introns from another gene. The sequences of the gene-targeting construct are arranged such that the foreign gene segment functions as the terminal set of exons for the targeted gene.

The codon reading-frame of the exons of the foreign gene segment can be arranged in-frame with the codon reading-frame of the nearest upstream (5') exon (exon 3 in this example) to form a complete open-reading frame. For proper splicing of messenger RNA to occur, a functional splice acceptor site immediately preceding the 5' exon of the foreign gene segment can be included. Thus, after splicing of the exon with the exons of the foreign gene segment, the transcript from the targeted gene will encode the desired gene product. The foreign gene segment can be obtained from various sources, as desired, and can be engineered to encode one or more mutations. The arrangement of a gene targeting vector is described above.

Another aspect of the present invention relates to host cells comprising a recombinant nucleic acid of the invention. A cell into which a nucleic acid is introduced is a transformed cell. Host cells include, mammalian cells, e.g., rodent, murine Ltk-, murine embryonic stem cells, COS-7, CHO, HeLa, insect cells, such as Sf9 and Drosophila, bacteria, such as E. coli,

Streptococcus, bacillus, yeast, fungal cells, plants, embryonic stem cells (e.g., mammalian, such as mouse or human), neuronal cells (primary or immortalized), e.g., NT-2, NT-2N, PC-12, SY-5Y, neuroblastoma. See, also Methods in Enzymology, Volume 185, ed., D.V. Goeddel. A nucleic acid can be introduced into the cell by any effective method including, e.g., calcium phosphate precipitation, electroporation, injection, DEAE-Dextran mediated transfection, fusion with liposomes, and viral transfection. When the recombinant nucleic acid is present in a host cell, it is preferably integrated by homologous recombination into a chromosome residing in the host cell.

The present invention also relates to a recombinant nucleic acid coding for a recombinant polypeptide, which nucleic acid is a product of the gene which has been modified by the gene targeting vector. A gene can code for different nucleic acid transcripts, depending on splicing, where it is expressed, etc. All such nucleic acids are a product of the recombinant gene and thus relate to the present invention. Such nucleic acids can code for recombinant polypeptides which are also an object of the present invention. The recombinant polypeptides can be used, e.g., as antigens to generate specific antibodies as diagnostic, research, and therapeutic tools.

A recombinant nucleic acid and a recombinant polypeptide can incorporate at least one amino acid or coding sequence thereof from a heterologous species. If, e.g., a non-human mammal sequence contains at least one amino acid of a human sequence, the modified sequence is described as "humanized. " By "humanized" it is meant, e.g., a mouse polypeptide containing one or more amino acids which are present in the human polypeptide (and which differ from the amino acids present in the mouse gene). Thus, in the examples, humanized mouse APP nucleic acids and polypeptides were created by substituting a human amino acid for a mouse amino acid at corresponding locations. A recombinant nucleic acid can be an unprocessed RNA transcript comprising introns or it can comprise a nucleotide sequence coding without interruption for amino acids, e.g., where the nucleic acid is a modified APP gene, it can code for amino acids 1-770,

1-713, 1-751, and 1-695. For example, a nucleic acid coding for a recombinant APP polypeptide can be a transcript from an APP gene modified in accordance with the present invention, e.g., by homologous recombination with a human cDNA and a mouse gene. The recombinant nucleic acid can comprise mutations in the APP gene, e.g., Swedish-FAD, London-FAD, etc., as described above.

The present invention also relates to a non-human transgenic animal, preferably a mammal, more preferably a rodent such as a mouse, which comprises a gene, which has been engineered employing a recombinant nucleic acid according to the present invention. Generally, a transformed host cell, preferably a totipotent cell, whose endogenous gene has been modified using a recombinant nucleic acid as described above is employed as a starting material for a transgenic embryo. The preferred methodology for constructing such a transgenic embryo involves transformed embryonic stem

(ES) cells employing a targeting vector comprising a recombinant nucleic acid according to the invention. A particular gene locus, e.g., APP, is modified by targeted homologous recombination in cultured ES cells employing a targeting vector comprising a recombinant nucleic acid according to the invention. The ES cells are cultured under conditions effective for homologous recombination. Effective conditions include any culture conditions which are suitable for achieving homologous recombination with the host cell chromosome, including effective temperatures, pH, medias, additives to the media in which the host cell is cultured (e.g., for selection, such as G418 and/or FIAU), cell densities, amounts of DNA, culture dishes, etc. Cells having integrated the targeting vector are selected by the appropriate marker gene present in the vector. After homologous recombination has been accomplished, the cells contain a chromosome having a recombinant gene. In a preferred embodiment, this recombinant gene contains host genomic sequences (e.g., mouse) fused to a donor cDNA (e.g., human). The cDNA can contain multiple mutations, etc., which are not naturally-occurring in the target gene. No further gene engineering steps are necessary. Thus, in accordance with the present invention, a single step has resulted in a modified gene containing as many modified sequences as desired. Another aspect of the present invention involves employing a cDNA with sufficient nucleotide sequence dissimilarity between it and the native target gene sequence to avoid inappropriate intra-recombination and inter- recombination events, subsequent to the first gene targeting step. The transformed or genetically modified cells can be used to generate transgenic non-human mammals, e.g., rodents (such as mice or rats), by injection into blastocysts and allowing the chimeric blastocysts to mature, following transfer into a pseudopregnant mother. See, e.g., Teratomacarcinoma and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed., IRL Press. Various stem cells can be used, as known in the art, e.g., AB-1, HM-1 D3, CC1.2, E-14T62a, preferably ES cell line Gl derived from inbred mouse strain 129/SvEvT.

In accordance with the present invention, a transformed cell contains a recombinant gene integrated into its chromosome at the targeted gene locus. A targeting vector which comprises sequences effective for homologous recombination at a particular gene locus, when introduced into a cell under appropriate conditions, will recombine with the homologous sequences at the gene locus, introducing a desired gene segment (e.g., a cDNA) into it. When recombination occurs such that insertion results, the nucleic acid is integrated into the gene locus. The gene locus can be the chromosomal locus which is characteristic of the species, or it can be a different locus, e.g., translocated to a different chromosomal position, on a supernumerary chromosome, on an engineered "chromosome," etc. In the examples described below, the sequences of the human APP gene are integrated by homologous recombination into the normal APP gene loci on murine chromosome 16. By recombinant, it is meant that the nucleotide sequences come from different sources, e.g., mouse and human.

A transgenic non-human mammal comprising a recombinant gene, which when mutant results in Alzheimer's disease, can express the gene in an amount effective to produce neuronal cell degeneration and/or apoptosis. The gene can also be expressed in an amount effective to cause a behavioral or cognitive dysfunction, wherein the dysfunction is conferred by the recombinant gene. Such gene can be, e.g., PS1, PS2, S182 (e.g., Sherrington et al., Nature, 375:754-760, 1995), STM2, E5-1, apoliprotein E, apoptosis genes such as ALG-1 to -6 (Vito et al., Science, 271:521, 1995), Bcl-2/Bax gene family, etc.

A transgenic non-human animal according to the present invention can comprise one or more genes which have been modified by genetic engineering. For example, a transgenic animal comprising an APP gene which has been modified by targeted homologous recombination in accordance with the present invention can comprise other mutations, including modifications at other gene loci and/or transgenes, including PS1, PS2, S182 (e.g., Sherrington et al., Nature, 375:754-760, 1995), STM2, E5- 1, apoliprotein E, apoptosis genes such as ALG-1 to -6 (Vito et al., Science,

111 : 521, 1995), Bcl-2/Bax gene family, etc. Modifications to these gene loci and/or introduction of transgenes can be accomplished in accordance with the methods of the present invention, or other methods as the skilled worker would know, e.g., by pronuclear injection of recombinant genes into pronuclei of one-cell embryos, incorporating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology. See, e.g., U.S. Patent Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci. , 77:7380-7384 (1980); Palmiter et al., Cell, 41:343-345 (1985); Palmiter et al., Ann. Rev. Genet. , 20:465-499 (1986);

Askew et al., Mol. Cell. Bio. , 13:4115-4124 (1993); Games et al. Nature, 373:523-527 (1995); Valancius and Smithies, Mol. Cell. Bio., 11: 1402-1408 (1991); Stacey et al., Mol. Cell. Bio. , 14: 1009-1016 (1994); Hasty et al., Nature, 350:243-246 (1995); Rubinstein et al., Nucl. Acid Res., 21:2613- 2617 (1993).

A recombinant nucleic acid molecule according to the present invention can be introduced into any non-human mammal, including a rodent, mouse (Hogan et al. , Manipulating the Mouse Embryo: A Laboratory

Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986), rat, pig (Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345, 1985), cattle or primate. See also, e.g., Church, Trends in Biotech. 5: 13-19, 1987; Clark et al., Trends in Biotech. 5:20-24, 1987; and DePamphilis et al., BioTechniques, 6:662-680, 1988.

A transgenic non-human animal and a recombinant nucleic acid molecule according to the present invention is useful as described in U.S. Pat. Nos. 5,304,489, 5,221,778, 5,175,385, 5,175,384, 5,175,383, 5,087,571, 5,082,779, 4,736,866, 4,873,191, and other transgenic animal patents. For example, a recombinant nucleic acid molecule comprising a coding sequence for at least one amino acid of a human APP gene is useful as a hybridization probe for detecting and diagnosing Alzheimer's disease, e.g., nucleotide variations and genetic polymorphisms present in a nucleic acid can be detected in accordance with various methods, e.g., U.S. Pat. 5,468,613; Conner et al., Proc. Natl. Acad. Sci. 80, 78 (1983); Angelini et al., Proc.

Natl. Acad. , 83, 4489 (1986); Myers et al., Science, 230, 1242 (1985). The nucleic acid can also be operably linked to an expression control sequence to produce polypeptide encoded by it. The operable linkage of nucleic acid and expression control sequence can be introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for the nucleic acid. An expression control sequence is similarly selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression. Other sequences which can be employed, include, enhancers such as from SV40, CMV, inducible promoters, neuronal specific elements, or sequences which allow selective or specific cell expression, such as in neuronal cells, glial cells, etc. The expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc. An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5' to a coding sequence, expression of the coding sequence is driven by the promoter. The resulting polypeptides can be used to generate antibodies for diagnostic purposes, etc. The operable linkage with an expression control sequence can also occur in situ as a result of homologous recombination at the desired gene locus, e.g., a mouse APP gene. A further aspect is the expression of a modified mRNA and polypeptides encoded by a recombinant nucleic acid molecule of the present invention in a transgenic animal, preferably a non-human mammal, as a model for diseases associated with the gene, e.g., the APP, PS1, and PS2 genes with Alzheimer's disease (AD), Down's syndrome, and heredity cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). Expression of a modified gene product in a transgenic non-human mammal and its consequent phenotype can therefore be used as a model for diseases and pathologies, e.g., as an AD model for genes associated with Alzheimer's disease. As described in the examples below, a mouse APP gene is modified by the introduction of mutations which are associated with an Alzheimer's phenotype in humans. Transgenic mice comprising cells which contain such a modified or recombinant APP gene can be used to design therapies. For example, active agents, e.g., synthetic, organic, inorganic, or nucleic acids based molecules, can be administered to a transgenic non-human mammal according to the present invention to identify agents which either inhibit, prevent, and/or reduce the appearance of an Aβ peptide in the brain, the AD pathology, neurodegeneration, apoptosis, cognitive deficits, and/or behavioral symptoms, etc. Thus, another aspect of the invention is to provide a method to assist in the advancement of the treatment and/or prevention of the aforementioned symptoms (e.g., neurodegeneration or apoptosis) caused by the APP gene, or a fragment thereof. Other genes and therapies can be used analogously.

Such a mammal model can also be used to assay for agents, e.g., zinc, and factors, e.g., environmental, which exacerbate and/or accelerate the diseases. See, e.g., Bush et al., Science, 265:1464-1467, 1994. A transgenic non-human animal can also be useful as pets, food sources (e.g., mice for snakes), in toxicity studies, etc.

Moreover, a non-human mammal containing a recombinant nucleic acid according to the present invention can be used in a method of screening a compound for its effect on a phenotype of a mammal, preferably a mouse, where the phenotype is conferred by the recombinant nucleic acid. By "phenotype," it is meant, e.g., a collection of morphological, physiological, biochemical, and behavioral traits possessed by a cell or organism that results from the interaction of the genotype and the environment. A phenotype can be behavioral, e.g., occurrence of seizures or cognitive performance, or it can be physiological and/or pathological, e.g., occurrence of neuronal cell degeneration, neuronal cell apoptosis, accumulation of Aβ peptide in the brain of the mammal, altered carboxy-terminal processing of the APP polypeptide, etc. According to such a method of detection, a compound can be administered to a mammal containing a modified APP gene and then the existence of an effect on the phenotype of the mammal can be determined. Observation can be accomplished by any means, depending on the specific phenotype which is being examined. For example, the ability of a test compound to suppress a behavioral phenotype can be detected by measuring the latter phenotype before and after administration of the test compound.

The invention also relates to a transgenic non-human mammal comprising cells that contain a recombinant gene modified by a gene targeting vector. For example, such recombinant gene or nucleic acid can code for a humanized mouse polypeptide comprising at least one amino acid coded for by a human gene, e.g., where the gene is the APP, PSl, or PS2 gene. In the case of the APP gene, the gene can code for, e.g., amino acids 1-665 of a mouse APP gene and amino acids 666-770 of a human APP gene, and having a phenotype conferred by the modified gene, e.g., accumulation of Aβ peptide or other related peptide in the brain, abnormal processing of the APP polypeptide, etc.

The level of expression of the recombinant gene can be any amount which can produce a phenotype in the non-human mammal, which phenotype can be distinguished from mammals which do not possess the modified gene locus, i.e., a control mammal, e.g., an amount effective to produce neuronal cell degeneration and/or apoptosis and/or an amount effective to cause a behavioral and/or cognitive effect or dysfunction where the gene is an alzheimer's disease associate gene. A non-human mammal containing a modified APP gene can also be characterized by accumulation of the Aβ peptide in its brain. The accumulation can be in any quantity which is greater than that observed in mammals not containing the modified gene locus. The phenotype conferred by the modified APP gene can occur before or after accumulation can be detected. The expression and/or accumulation of the APP polypeptide, and its processed derivatives, and the nucleic acids which encode it, can be measured conventionally, e.g., by immunoassay or nucleic acid hybridization, either in situ or from nucleic acid isolated from host tissues. The identification of agents which prevent and/or treat symptoms associated with expression of the modified gene can be determined routinely. For example, an active agent can be administered to a transgenic mammal comprising a modified gene according to the present invention and then its effect on a behavior or pathology, e.g., Aβ deposition in the brain, apoptosis, and/or neurodegeneration, can be determined. The agent can be administered acutely (e.g., once or twice) or chronically by any desired route, e.g., subcutaneously, intravenously, transdermally, or intracathically. The formulation of the agent is conventional, see, e.g., Remington's Pharmaceutical Sciences, Eighteenth Edition, Mack Publishing Company,

1990. In a test, e.g., an agent can be administered in different doses to separate groups of transgenic mammals to establish a dose-response curve to select an effective amount of the active agent. Such effective amount can be extrapolated to other mammals, including humans. The transgenic mammal, preferably a mouse, according to the present invention therefore permits the testing of a wide variety of agents and therapies. In AD, for example, a number of different agents have been identified which affect the cognitive dysfunction associated with the diseases, e.g., cholinergic agents, such as muscarine agonists, acetylcholinesterase inhibitors, acetylcholine precursors, biogenic amines, nootropics, angiotensin converting enzyme (ACE), and vitamin E. In addition, agents which regulate APP or Aβ expression, Aβ deposition, and physiological changes associated with Aβ expression and deposition can also be identified, e.g., calcium homeostasis, inflammation, neurofibrillary tangles. See, e.g., Pavia et al., Annual Reports of Medicinal Chemistry, 25:2129, 1989; John et al., Annual

Reports of Medicinal Chemistry, 28:197-203, 1993. Additionally, active agents which block apoptosis, e.g., free radical scavengers, such as glutathionines, can be administered. Such effects on AD can be assayed in either behavioral or physiological and/or histological studies. For example, spatial learning and memory abilities in mice can be tested in a Morris water maze. See, e.g., Yamaguchi et al., NeuroReport, Vol. 2, 781-784 (1991). Additionally, other behavioral tests can be used, e.g., Swim Test, Morris et al., Learning and Motivation, 12, 239-260, 1981; Open-field test, Knardahl et al., Behav. Neurol. Biol. 27, 187-200, 1979; and tests and models used routinely, e.g., in mice, rats, and other rodents.

According to the present invention, differences in, e.g., levels of expression, cellular localization, and/or onset of expression of the recombinant gene can be used to model a disease, e.g., AD and other diseases associated with APP expression and the differing stages and progressions of the disease, e.g., cell degeneration, cell death, astrogliosis, and/or amyloidosis. Having a range of expression phenotypes can be useful to identify different therapies and drug treatments and also diagnostically to identify a disease's progression. For example, the specific treatments can depend on the region of the brain in which an APP peptide is expressed, how much of it is expressed, and its temporal progression of expression. Thus, mammals having different phenotypes can be used as models for determining therapies which are selective for different stages of the disease and for studying disease progression and intervention.

DESCRIPTION OF THE FIGURES

Figure 1. Schematic of p35A; mouse APP exon 16 genomic clone

The ^"15 Kb Not I genomic fragment (shown) was isolated from the lambda clone 35 A and cloned into the Not I site of Bluescript II SK+ . Exon 16 is indicated and begins approximately 9.5 Kb from the 5' -end of the genomic fragment. The indicated restriction enzyme recognition sites were placed for reference.

Figure 2. Restriction Map pMTI-2396 pMTI-2396 contains mouse APP exon 16 and was derived from the ^"5.5 Kb Ncol fragment from p35A (Ncol at position 7645 to Ncol at position

13176, Figure 1). The 5.5 Kb Ncol fragment was inserted into Ncol- modified Bluescript II SK+ at the Ncol site. All recognition sites for the indicated restriction enzymes are designated. Sequence from positions 29 to 5560 were derived from the mouse APP gene and the remaining sequences were derived from Bluescript II SK+ .

Figure 3. Restriction map of pRA3 pRA3 contains mouse APP intron 15 sequences and was derived from the ^"3 Kb Ncol fragment from p35A (Ncol at position 4816 to Ncol at position 7645, Figure 1). The 3 Kb fragment was inserted into Ncol- modified Bluescript II SK+ at the Ncol site. All recognition sites for the indicated restriction enzymes are designated. Sequence from positions 29 to 2858 were derived from the mouse APP gene and the remaining sequences were derived from Bluescript II SK+ .

Figure 4. Restriction map of pN2C4 pN2C4 contains mouse APP intron 16 sequences and was derived from the "1.9 Kb Ncol fragment from p35A (Ncol at position 13176 to Ncol at position 14992, Figure 1). The ^"1.9 Kb fragment was inserted into Ncol- modified Bluescript II SK+ at the Ncol site. All recognition sites for the indicated restriction enzymes are designated. Sequence from positions 29 to

1845 were derived from the mouse APP gene and the remaining sequences were derived from Bluescript II SK+ .

Figure 5. Restriction map of pMTI-2398; Swedish-FAD targeting vector

The mouse APP intron 15 and exon 16 sequences encompass positions 30 to 1960 (Bglll site). The human APP cDNA and genomic polyadenylation sequences are contained in sequences between positions 1960 and ^"4556.

The neomycin resistance gene lies between positions ^"4556 and ^"6460.

Mouse APP intron 16 sequences are contained between positions ^"6460 and

9872. The Bluescript II SK+ sequences are between positions ^"9872 and ^"30. All recognition sites for the indicated restriction enzymes are designated.

Figure 6. Restriction map of pMTI-2453; London-FAD targeting vector The HSV TK gene is located between positions ^"17 and ^"2893. The mouse APP intron 15 and exon 16 sequences encompass positions "2906 to 4835 (Bglll site). The human APP cDNA and genomic polyadenylation sequences are contained in sequences between positions 4835 and ^"7452.

The neomycin resistance gene lies between positions ^"7452 and "9323.

Mouse APP intron 16 sequences are contained between positions ^"9323 and

12750. The Bluescript SK+II sequences are between positions ^"12750 and ^"37. All recognition sites for the indicated restriction enzymes are designated. Figure 7. Restriction map of pMTI-2454; Swedish/London-FAD targeting vector

The HSV TK gene is located between positions ^"17 and ^"2893. The mouse APP intron 15 and exon 16 sequences encompass positions ^"2906 to 4835 (Bglll site). The human APP cDNA and genomic polyadenylation sequences are contained in sequences between positions 4835 and ^"7452.

The neomycin resistance gene lies between positions ^"7452 and ^"9323.

Mouse APP intron 16 sequences are contained between positions ^"9323 and

12750. The Bluescript II SK+ sequences are between positions ^"12750 and "37. All recognition sites for the indicated restriction enzymes are designated.

Figure 8. Restriction map of pMTI-2455 (Swedish-FAD APP713 targeting vector)

The HSV TK gene is located between positions ^"17 and ^"2893. The mouse APP intron 15 and exon 16 sequences encompass positions ^"2906 to

4835 (Bglll site). The human APP cDNA and genomic polyadenylation sequences are contained in sequences between positions 4835 and ^"7452.

The neomycin resistance gene lies between positions ^"7452 and ^"9323.

Mouse APP intron 16 sequences are contained between positions ^"9323 and 12750. The Bluescript II SK+ sequences are between positions "12750 and

^"37. All recognition sites for the indicated restriction enzymes are designated.

Figure 9. Oligonucleotides

Oligonucleotides are designated in the 5' to 3' direction.

Figure 10. Schematic outline of m/hAPP gene products produced in transgenic mouse lines. The protein m/hAPP exhibits amino acid sequence identity with mouse APP with the exception of those residues indicated by (asterisks, see text above). m/hAPP protein spans the membrane once as indicated. The bA4 peptide region (indicated by red) partially resides in the transmembrane and extracellular domains. The APP751 alternative splice form of APP has the

56 amino acid Kunitz protease inhibitor domain while the APP770 splice form of the protein has both the Kunitz and the 19 amino acid OX domains. The APP695 alternative splice form of APP contains neither Kunitz nor OX domains. Other splice forms are not indicated. There are two possible N- linked glycosylation sites (CHO) in the extracellular domain of APP. A highly negatively-charged domain and a cysteine-rich domain are symbolized by a minus sign and S-S bridges respectively. The signal peptide (SP) is located at the N-terminus (see Unterbeck et al.).

Figure 11. Gene-targeting strategy: Construction of targeting vectors. The schematic of the Nco I APP gene fragment represents the "5.5 Kb

Ncol mouse APP gene fragment in pMTI-2396 (Figure 2). The regions indicated in red represent the coding sequences for mouse b-amyloid domain. The schematic for the targeting vector represents the linearized (using Ascl) DNA from clone pMTI-2454 (Figure 7). The targeting vectors for pMTI- 2453 (Figure 6) and pMTI-2455 (Figure 8) are identical to pMTI-2454 with the exception of the FAD mutation and the orientation of the HSV TK gene (see text). pMTI-2398 is similar to pMTI-2454 with the exception of FAD mutation and the absence of the HSV TK gene (see text). The FAD mutations are indicated by black asterisks and the mutations to "humanize" the b-amyloid domain are indicated by green asterisks. The neomycin resistance gene is designated by neo^r and Bluescript II SK+ sequences are designated by BSSK+ . Figure 12. Gene-targeting strategy: Homologous recombination.

The linearized targeting vector (Figure 11) was electroporated into ES cells. Homologous recombination occurred between mouse APP sequences contained in the targeting vector and mouse APP genomic sequences on chromosome 16. The resulting targeted m/hAPP gene locus is schematically shown. The FAD mutations are indicated by asterisks and the mutations to "humanize" the b-amyloid domain are indicated by asterisks.

Figure 13. Gene-targeting strategy: Targeted m/hAPP gene locus.

The comparison of the mouse APP and targeted m/hAPP gene loci is shown schematically. In normal mouse, the b-amyloid, transmembrane, and cytoplasmic domains of APP are encoded by mouse APP exons 16, 17, and 18. In the case of the targeted m/hAPP gene locus, the b-amyloid, transmembrane, and cytoplasmic domains of m/hAPP are encoded by human cDNA sequences. The remainder of m/hAPP is encoded by mouse APP exons 1 through 15. The FAD mutations are indicated by asterisks and the mutations to "humanize" the b-amyloid domain are indicated by asterisks.

Figure 14. Strategy for Southern-blot detection of ES cells having a targeted m/hAPP gene locus containing the Swedish-FAD mutation (e.g.; transgenic lines ES5007 and ES5103) .

The schematics for the mouse and m/hAPP loci are indicated. The restriction enzymes Xbal and Ncol are designated by X and N respectively. The box represents human APP cDNA and genomic sequences while the box represents the neomycin resistance gene.

Figure 15. Gene Targeting Strategies

A. Normal Gene B. Targeted gene. Fusion of a gene with cDNA (in-frame fusion of mouse exon sequences with cDNA). * represents one or more mutations.

C. Targeted gene. Fusion of a target gene with cDNA (cDNA is inserted into a mouse intron (intron 3 for example). The cDNA is directly preceded by a splice acceptor site. The sequence of the insert is formatted so that splicing of the 3 '-sequence of the exon (exon 3 for example) with the 5'- sequence of the cDNA will create a mature transcript encoding the appropriate gene product). * represents one or more mutations.

D. Targeted gene. Fusion of a targeted gene with a foreign (same or different species) gene segment including one or more exons inserted into the intron of the targeted gene. The sequence of the insert is formatted so that splicing of the 3 '-sequence of the mouse exon (exon 3 for example) with the 5 '-sequence of the other mouse gene or species exon (exon 4' for example) will create a mature transcript encoding the appropriate gene product). * represents one or more mutations.

Figure 16. Amino acid sequence of human APP.

Figure 17. Sequence of mouse exon 16 locus

Figure 18. Sequence of pMTI-2398 (Swedish-FAD APP targeting vector )

Figure 19. Sequence of pMTI-2453 (London-FAD APP targeting vector)

Figure 20. Sequence of pMTI-2454 (Swedish/London-FAD APP targeting vector)

Figure 21. Sequence of pMTI-2455 (Swedish-FAD APP713 targeting vector) Figure 22. Sequence of APP genomic clone containing human APP polyadenylation signals.

EXAMPLES

Four independent lines of transgenic mice (lines ES5007, ES5103, ES5401 and ES5403) have been created via a novel gene targeting technique applied to embryonic stem cells. In each line, the mouse APP gene has been modified to encode a mouse/human hybrid APP (m/hAPP) where amino acid residues 666-770 of APP770 are now encoded by human cDNA sequences instead of mouse genomic exons (exons 16, 17, and 18). Within these residues only three amino acid differences exist between the mouse and human proteins (Gly (676) to Arg, Phe(681) to Thr, and Arg(684) to His). This exon-cDNA fusion gene, therefore, encodes an APP containing a

"humanized" beta-amyloid domain (aa residues 672 to 712).

In each transgenic mouse line, the human cDNA sequences have been modified to introduce one or more mutations proximal to the "humanized" beta-amyloid domain. In transgenic mouse line ES5007, m/hAPP has been mutated to include the "Swedish"-FAD mutation (KM to NL, positions 670 and 671)(Cai et al., 1993, Citron et al., 1994). Transgenic mouse lines ES5401 and ES5403 encode m/hAPP which have been mutated to include the "London"-FAD mutation (V to I, position 717) (Suzuki et al., 1994, Gravina, 1995). Transgenic mouse line ES5103 encodes m/hAPP which has been mutated to include both "London" and "Swedish" FAD mutations. A fifth transgenic mouse line ES5215 can be produced which encodes m/hAPP that has been mutated to include both the "Swedish" FAD mutation and a premature stop codon (T to stop at position 714). With the exception of the changes mentioned above, the remainder of the m/hAPP sequences are identical to those found in normal mouse APP.

We have shown that the targeted Swedish-FAD m/hAPP and Swedish/London-FAD m/hAPP genes express m/hAPP protein at levels approaching those observed for mouse APP in brain. Notably, we have observed that the Swedish FAD mutation alters significantly the proteolytic processing of APP resulting in differences in the appearance of C-terminal fragments. The observed changes in processing is consistent with the Swedish-FAD mutation inducing the beta-secretase cleavage site to be utilized predominately over the alpha-secretase cleavage site as previously observed in cell culture experiments (see below).

Messenger RNA from the Swedish-FAD m/hAPP gene was found be abundantly expressed in the brain from homozygous ES5007 mice as well. The amount of Swedish-FAD m/hAPP mRNA in homozygous ES5007 brain was determined to be approximately 55% of the mAPP mRNA levels observed in control mouse brain. In concordance, the APP mRNA levels in heterozygous ES5007 mouse brain were found to be approximately 75 % of the level observed in control mouse brain.

The reverse transcriptase-PCR (rtPCR) technique was used to identify mouse APP and Swedish-FAD m/hAPP transcripts in mouse brain.

Homozygous ES5007 mice were found to express mRNA exclusively from the targeted Swedish-FAD m/hAPP gene. No mRNA species containing sequences from mouse APP exons 16, 17, or 18 was detected in homozygotes. As would be expected, heterozygous ES5007 mice were found to express mRNA transcripts from both normal mouse and Swedish-FAD

APP alleles.

Western-blot analyses have demonstrated that Swedish-FAD m/hAPP and Swedish/London-FAD protein is expressed in the brain of ES5007 and ES5130 mice, respectively. Swedish-FAD m/hAPP protein is expressed in the brain of homozygous ES5007 mice at approximately 87% of the level observed for mouse APP in non-transgenic mice (n = 4). Retrieving mouse APP exon 16 from genomic library

Phage lifts: The mouse 129 genomic library from Stratagene (cat#946308) was titered and plated out 20 150 mm LB plates containing "50,000 phage/plate. Duplicate lifts were made from each plate using Amersham Hybond-N-f- nylon membranes. The plates were refrigerated for several hours to ensure the top agar was hardened. The membranes were placed atop the plaques and left on for 5 minutes. The membranes were lifted off the plates and placed plaque-side up on 3MM paper saturated with denaturation solution (0.1 M NaOH, 1.5 M NaCl) for 5 minutes. The membranes were transferred briefly to dry 3MM paper to absorb the excess solution and then placed on 3MM paper saturated with neutralizing solution (0.2 M Tris-Cl pH 7.5, 2X SSC) for 5 minutes. The membranes were rinsed by placing them on 3MM paper saturated with 2X SSC for 5 minutes and then air dried. A digoxigenin-labeled mouse specific APP exon 16 probe of 93 bp was generated using PCR (from nt 1877 to 1969 in sequence

MUSABPPA, accession #M 18373).

PCR assay: In a 50 μl total reaction volume was added 1 μg genomic mouse tail DNA, 5 μl 10X PCR buffer (Perkin Elmer cat#N808-0006), 5 μl 2 mM dATP, dCTP, dGTP mix, 5 μl 1.3 mM dTTP, 3.5 μl 1 mM digoxigenin-11-dUTP, 3 μl 100 ng/ml oligonucleotide mix of KC65

(5'GTTCTGGGCTGACAAACATC3') and KC66

(5'GATGGCGGACTTCAAATCCTG3'), and 2.5 units AmpliTaq (Perkin Elmer cat#N808-0070). The reaction was run in a Perkin Elmer turbo 9600 thermal cycler. The parameters of the run were as follows: one cycle at 94°C for one minute, 36 cycles at 94°C for 30 seconds-56°C for 50 seconds-

70 °C for two minutes, maintain at 10°C indefinitely. Four individual PCR reactions were pooled and passed through a Sephadex G-50 column from Boehringer-Mannheim (cat#100616) in 10 mM Tris-Cl pH 7.5, 1 mM EDTA, and 0.1 % SDS. Several dilutions of the dig-labeled probe were blotted onto a membrane and compared to standard amounts of a dig-labeled control DNA.

Hybridization of plaque-lifted membranes: Membranes were pre- hybridized in 50% formamide, 5X SSC, 0.1 % N-lauryl sarcosine, 0.02%

SDS, and 5% blocking reagent supplied by Boehringer-Mannheim (cat# 1096176) and incubated at 42 °C for 4 hours. The pre-hybridization solution was discarded and replaced with identical fresh hybridization solution that contained 2 μg of the dig-labeled mouse APP exon 16 probe that was boiled for 10 minutes and chilled on ice. Membranes were hybridized over a two-day period at 42 °C. All incubations (and heated-washes) were performed in the Stovall "Belling Dancing" water bath. The probe/hybridization solution was removed and saved for subsequent screenings. Membranes were washed four times in 2X SSC, 0.1 % SDS at room temperature for 5 minutes. Subsequent washings were as follows: two washes of 30 minutes at 65 °C in 0.5X SSC, 0.1 % SDS; two washes for 30 minutes at 65 °C in 0.2X SSC, 0.1 % SDS; ten minutes at 65 °C in 0.2X SSC; and ten minutes at room temperature in 0.2X SSC.

Digoxigenin detection assay: The remaining protocol is taken from the Boehringer-Mannheim "DIG Nucleic Acid Detection Kit " (cat# 1175041 ) .

Membranes were rinsed once for 2 minutes at room temperature in Genius 1 buffer (100 mM Tris-Cl, pH 7.5, 150 mM NaCl) and blocked for 1 hour at room temperature in Genius 2 buffer (2% w/v blocking agent in Genius 1 buffer). Membranes were incubated with 150 μunits/ml of polyclonal sheep anti-digoxigenin alkaline phosphatase conjugated antibody in Genius 2 buffer for 30 minutes at room temperature. Two washes were done for 15 minutes each at room temperature in Genius 1 buffer and once for 2 minutes in AP 9.5 buffer (100 mM Tris-Cl pH 9.5, 100 mM NaCl, 50 mM MgCl₂). Membranes were processed in Lumi-Phos 530 (Boehringer-Mannheim cat# 1275470) and placed in the dark for 16 hours then exposed to film for 20 minutes. Positive plaques were picked and placed into 1 ml SM buffer (5.8 g

NaCl, 2.0 g MgS0₄-7H₂0, 50 ml 1 M Tris-HCl pH 7.5 to a total volume of one liter) to diffuse and stored at 4°C. These plaques were screened in a PCR assay using the identical oligonucleotide pair that was used to generate the probe (assay- 15 μl phage stock and 35 μl water were heated to 95 °C for 20 minutes into which was added 10 μl 10X PCR buffer, 3 ml 100 ng/ml oligo mix of KC65 and KC66, 10 μl 2 mM dNTP mix, 5 units AmpliTaq, and 1 unit Perfect Match Polymerase Enhancer (Stratagene cat# 600129) to a total volume of 100 μl).

Secondary membrane screenings on 4 isolates were performed using the digoxigenin-mouse APP exon 16 probe previously made. Two positive phage plaques were grown (protocol taken from BioTechniques 7:21-23) to obtain enough DNA for further analysis.

A 15 Kb sequence containing the mouse APP exon 16 was sub-cloned into pBluescript IISK-f- (Stratagene cat#212205) at the Notl site (designated as plasmid 35 A) using standard cloning procedures. Southern analysis using a 32p-labeled mouse APP exon 16 probe revealed a 5 Kb Ncol fragment which became the backbone into which our human APP cDNAs were fused.

Southern analysis: Six separate reactions containing 1 μg of plasmid 35 A were digested with 10 units each of restriction enzymes Apal (cat#114S), Apal/Bglll (cat#144L), Ncol (cat#193L), Ncol/Bglll, Xbal (cat#145S),

Xbal/Bglll (supplied by New England Biolabs) in their respective buffers (total volume of 30 μl) at their respective incubation temperatures for 3 hours. One-half of the digestion reactions was loaded onto an 0.8% agarose (Bio-Rad cat#162-0133) gel in IX TBE buffer. The gel was run at 20 volts overnight at room temperature. After photographing the gel, it was prepared for transferring to a nylon membrane. The gel was soaked in 0.25N HC1, rocked gently, for 15 minutes, rinsed well with water then soaked in 0.4N NaOH, rocked gently, for 20 minutes. A 3MM paper wick transfer was assembled using Amersham Hybond-N+ nylon membrane in 0.4N NaOH buffer overnight at room temperature. The membrane was rinsed in 5X SSC for 10 minutes at room temperature and UV cross-linked in a Stratalinker (Stratagene cat. #400071) using 1.2xl0⁵ mjoules for 30 seconds. The membrane was hybridized in 50% deionized formamide, 5X SSC, 0.1 % N- lauryl sarcosine, 0.02% SDS, and 5% blocking agent (Boehringer- Mannheim) at 42 °C, rocked gently, and incubated overnight. This solution was removed and replaced with the previously made mouse APP exon 16 digoxigenin-labeled probe (denatured) in fresh hybridization buffer and incubated at 42 °C, rocked gently, for overnight. All subsequent washes, blocking, and antibody binding was identical to the protocol stated previously as digoxigenin detection assay.

Construction of the targeting vectors

Subcloning mouse exon 16 locus: The 5 Kb Ncol fragment containing the mouse APP exon 16 sequence was cloned into pBluescript IISK+ at an engineered Ncol site to generate pMTI2396 (Figure 2; see below). The 3 Kb 5 '-flanking Ncol fragment and 2 Kb 3 '-flanking Ncol fragments from p35A were also cloned into pBluescript IISK+ at the engineered Ncol site to generate pRA3 and pN2C4, respectively (Figures 3 and 4; see below). The pBluescript vector (1 μg) was digested with 20 units of Xbal in buffer 2

(NEB) and incubated for 2 hours at 37 °C. Ten units of calf intestine alkaline phosphatase (CIP from Boehringer-Mannheim cat#713023) were added to the reaction and incubated for 1 hour at 37 °C to dephosphorylate the 5' ends. To 500 ng of the 5Kb Ncol fragment was added 6.2 pmol of phosphorylated, annealed adapter KC95/96 (5'CTAGACACTC3') using 400 units of T4 DNA Ligase (NEB cat#202L) in its appropriate buffer (50 mM Tris-Cl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP 25 mg/ml BSA) at 25 °C for a 5-hour incubation. This reaction was digested with 20 units of Xbal and adjusted the buffer concentration to 50 mM NaCl and incubated for 1 hour at 37 °C. The enzyme was heat inactivated at 65 °C for 20 minutes. The DNA was removed from the residing enzymes using Strataclean resin (Stratagene cat#400714). To the 25 μl enzyme digestion reaction was added 5 μl of Strataclean resin, vortexed for 15 seconds and set at room temperature for 1 minute. It was then spun in an Eppendorf microcentrifuge 5415C at 14000xg for 1 minute. The supernatant was transferred to a clean tube and the procedure was repeated once. Dephosphorylated Xbal-linearized pBluescript, 50 ng, was combined with 500 ng of the phosphorylated 5Kb Ncol-adapter fragment in a standard ligation reaction and incubated at 14°C for overnight. The ligase was heat inactivated at 70 °C for 10 minutes and one-tenth of the reaction was transformed into Epicurian.coli XL-1 blue cells (Stratagene cat#200236) using the protocol provided. The resulting construct having mouse genomic sequences for the 3' end of intron 15-exon 16-5' end of intron 16 was then referred to as p2396 (Figure 2). The Bglll site within exon 16 is the point at which the human cDNA sequence was fused.

Subcloning of Ncol fragments proximal to the Exon 16 targeting site: The 3 Kb intron 15 fragment and the 2 Kb intron 16 fragment were generated by a Ncol digestion on the template plasmid 35 A (see Figure 1). The 3Kb and 2 Kb Ncol-Ncol fragments were then subcloned into the Bluescript

(Ncol) vector (see above). The resulting plasmids were named pRA3 (3 Kb fragment; Figure 3) and pN2C4 (2Kb fragment; Figure 4). They were expanded and the 2 and 3 Kb fragments themselves isolated by Geneclean (Bio 101). These isolated fragments were then used as probes in Southern blot paradigms.

Generation of cloning sites around the neomycin resistance gene: As an integral part of our targeting vector construct, we cloned the neomycin resistance gene (pPol21ongneobpA provided by Ann Davis) downstream of our human APP cDNA sequence. The neomycin resistance gene (contained within a pBluescript KS + vector) was under transcriptional regulation of the DNA polymerase II promoter sequence (long version) and the bovine growth hormone (BGH) polyadenylation sequences. Sequences composed of different restriction sites had to be cloned onto both the 5 ' and 3 ' ends of this gene construct. The plasmid, 2 μg, was linearized with Sail, ligated to 45 pmol of annealed Sall-Aflll-EcoRV-NcoI-MluI adapter (5 ' TCGACGACTT AAGTTGATATCC ACC ATGGTGACGCGTT3 ' ) using 400 umts of T4 DNA Ligase in its appropriate buffer at 14 °C in an overnight incubation. This reaction was digested with EcoRV (cat#195S) and ligated to close. This plasmid, now referred to as p2395, was digested with Xhol to linearize it at the 3' end of the BGH sequence. Ligated to this Xhol site was an Xhol-Bglll-Stul adapter (5 CGAGTGAGATCTTAAGGCCTGG3'). The ligase was removed from the reaction using the Wizard DNA clean up system (Promega cat#A7280) following the directions supplied in the kit. The linearized plasmid-adapter DNA (approx. 5 μg) was digested with 30 units each, in one 50 μl reaction, of Stul (cat#187L)/EcoRV in restriction enzyme buffer 2 (from NEB) at 37 °C for 3 hours. The digest reaction was run through a 0.8% low melt agarose (FMC cat#50112) gel in 0.5X TAE buffer (20 mM Tris acetate, 0.5 mM EDTA) at 75V for 2 hours at room temperature. The 1800 bp band containing the promoter/neomycin/polyA sequences was excised from the gel and extracted from the agarose using the Wizard DNA clean up system. This fragment was ligated to the human APP cDNA-adapter generated through the follow process.

Generation of human APP cDNA's with either the Swedish-FAD. London-FAD. Swedish/London-FAD_f or Swedish-FAD. APP713 mutations fused with human APP genomic sequences containing APP polyadenylation signals: Plasmid pMTI-2385-Swedish (not shown) possesses the entire human APP 695 cDNA fused with human APP cloned into pBluescript II SK+ . The plasmid pMTI 2398 was derived from pMTI2385. The strategy for its creation involved the extensive use of a cDNA-genomic hybrid plasmid, pMTI2339. pMTI2385-Swedish was assembled in a four-part ligation with the following components; an ^"1861 bps. Xmal-Bglll fragment from pMTI2339, a ^"2008 bps. Spel-Sall fragment from pMTI2339, a ^"589 bps. fragment from FAD clone #5 (contains Swedish-FAD mutation) generated by Dr. Gerhard Konig, and a pBSSK(+)II vector opened up with Xmal and Sail. The ligation was done according to standard protocols with the insert fragments being in equal molar ratios and there being a 3:1 ratio of total insert to vector. Ligation mixtures were transformed in XL-1 Blue competent cells (Stratagene) and mini-preps analyzed by an initial digestion of Xmal-Sall. Two putative clones were further characterized with Bglll-Spel, Xmal-Bglll, Spel-Sall, EcoRI, Hindi, and PvuII. Two clones, #4 and #5 gave the expected results. These were grown up and sequenced confirmed. Plasmid pMTI-2453 was derived from pMTI-2385-London. pMTI- 2385-London was assembled in a four-part ligation with the following components: a ^"1.7 Kb Xma I-Sacl fragment from pMTI2385-Swedish, a ^"350 bp Sacl-Styl fragment from pMTI-104 (contains London-FAD mutation; obtained from Paul Fracasso), a ^"2.5 Kb Styl-Sall fragment from pMTI2385- Swedish, and a ^"2.7 kb Sall-Xmal fragment from pMTI2385-Swedish. Plasmid pMTI-2454 was derived from pMTI-2385-Swedish/London. Swedish/London was assembled in a four-part ligation with the following components: a "1.9 Kb Xmal-EcoRI fragment from pMTI2385-Swedish, a "700 bp EcoRI-Clal fragment from pMTI-2385-London, a ^"1.9 Kb Clal-Sall fragment from ρMTI2385-Swedish, and a^"2.7 Kb Sall-Xmal fragment from pMTI2385-Swedish.

Plasmid pMTI-2455 was derived from pMTI-2385-Swedish APP713. pMTI-2385-Swedish APP713 was assembled in multi-step process using PCR mutagenesis to introduce the APP713 stop mutation into proximity with the Swedish-FAD mutation. First, a ^"560 bp EcoRI-Spel fragment from pMTI2385-Swedish was ligated with the 2.9 Kb EcoRI-Spel fragment from Bluescript KS+II (Stratagene) to generate pMTI-X. A ^"400 bp fragment containing the APP 713 stop mutation was generated by PCR using APP cDNA as template and oligonucleotides RA39 (CCATCGATGGATCAGTTACGGAAACGATGCTCTCATGC) and RA40

(CCATCGATGGCCAAGGTGATGACGATCACTGTGGATCCCTACGCT ATGACAACACCGC) (Figure 9). The ^"400 bp PCR fragment was digested with Clal and Sty I and ligated into the ^"3.3 Kb Clal-Styl fragment from pMTI-X to generate pMTI-Y. pMTI-2385-Swedish APP713 was assembled in a four-part ligation with the following components: a ^"560 bp EcoRI-Spel fragment from pMTI-Y, a ^"1.9 Kb Xmal-EcoRI fragment from pMTI2385- Swedish, a ^"2 Kb Kb Spel-Sall fragment from pMTI2339, and a ^"2.8 Kb fragment from Bluescript SK+II.

Generation of the human APP "Swedish" FAD mutation cDNA- neomycin sequences to fuse to the mouse APP exon 16 DNA: Four μg of plasmid pMTI-2385B was digested with 20 units of restriction enzyme Sail (cat#138L) in its ideal buffer for 2 hours at 37 °C. The reaction was run through an 0.8% agarose gel in 0.5X TAE buffer at 120 volts for 1.5 hours at room temperature. The linearized DNA band was excised and isolated away from the agarose using the Qiaex DNA Gel Extraction kit and the protocol provided (Qiagen Cat#20021 ). One μg of Sall-linearized p2385B was ligated to 45 pmol of annealed Sall-Aflll-EcoRV-NcoI-MluI adapter (mentioned previously) in a standard ligation reaction. One-tenth of the ligation reaction was used to transform E.coli XL-1 blue cells. This p2385B-adaptor construct, 18 μg, was linearized with 60 units of EcoRV in a standard digestion reaction. Into this was added 15 units of calf intestine alkaline phosphatase and incubated at 37 °C for 1 hour to dephosphorylate the 5' ends of the DNA. The reaction was stopped with EDTA at a final concentration of

5 mM and heat inactivated at 75 °C for 10 minutes. The dephosphorylated plasmid was gel isolated and 1 μg was ligated to 400 ng of the 1800 bp neomycin fragment with EcoRV 5' and Stul 3' ends (mentioned in the section "Generation of cloning sites around the neomycin resistance gene"). One- tenth of the ligation reaction was used to transform E. coli XL-1 blue cells following the protocol provided by the supplier. Correct orientation constructs had the neomycin fragment (5' EcoRV site) placed immediately downstream of the human APP cDNA poly A sequences (3' EcoRV site), this construct was designated p2397+A (not shown).

Construction of the completed targeting vector containing the human

APP "Swedish" FAD mutation: The 5 Kb mouse APP exon 16 containing DNA, p2396 (12 μg), was digested with 50 units of Bglll in buffer 3 for 3 hours at 37 °C. To 6 μg of the digest was added 10 units of CIP and incubated at 37 °C for 1 hour. The reaction was stopped as mentioned above and the DNA was gel isolated using Gelase (Epicentre cat#G09100) and following the supplied protocol. The 4.5 Kb Bglll fragment containing the human APP cDNA-neomycin fusion was released from p2397+A by digesting 12 μg of DNA with 28 units of Nrul (cat#192L) in its ideal buffer at 37 °C for 3 hours. After being confirmed of its linearization 50 units of Bglll were added for an additional 2-hour incubation at 37 °C. The 4.5 Kb fragment was gel isolated using Gelase and then ligated (300 ng) to the dephosphorylated p2396-BglII linearized DNA (100 ng) in a standard ligation reaction and subsequently transformed into E.coli XL-1 blue cells. The resulting plasmid with the mouse APP exon 16 fused to the human APP cDNA at exon 16 (Bglll site) was designated as p2398 (Figure 5 and Figure 18).

E.2.7 Cloning of the HSV thimidine kinase (TK) gene into the targeting vector: The HSV thimidine kinase gene (from pAD7) was provided by Ann Davis. Unique restriction sites had to be engineered with the TK gene to provide linearizing access in the completed targeting vector. A 3Kb BamHI-Clal fragment containing the murine phosphoglycerate kinase (PGK) promoter regulating the TK gene with the BGH polyadenylation sequences was isolated away from vector sequences and sub-cloned into pBluescript II

SK+ at its respective sites. Twenty μg of this new TK plasmid, pCBl 1 , was digested with 60 units of Sail in its unique buffer and incubated overnight at 37 °C. The enzyme was heat inactivated at 65 °C for 20 minutes and then 10 units of CIP was added for 1 hour at 37 °C. The phosphatase was heat inactivated at 75 °C for 10 minutes. The linearized DNA band was excised and isolated away from the agarose using the Qiaex DNA Gel Extraction kit and the protocol provided as stated above. In a standard ligation reaction, 45 ng of Sall-linearized vector was added to 15 pmol of annealed Sall-Ascl- Pmel-Notl-Ascl-Pmel-Sall adapter (5 ^* TCGAC AAGGCGCGCCGTTTAAAC AAGCGGCCGCTTGGCGCGCCT

TTTGTTTAAACTTG3') and incubated overnight at 14°C. This TK plasmid containing the restriction sites Pmel and Ascl was designated as pXII28N. Five μg of pXII28N was digested with 20 units of Notl (cat#189L) and 15 units of Pvul (cat#150L) in Notl buffer (NEB) and 0.1 mg/ml BSA at 37 °C for overnight. The 3Kb Notl TK band was excised and isolated away from the agarose using the Qiaex DNA Gel Extraction kit and the protocol provided. Five hundred ng of this TK fragment was ligated to 50 ng of Notl linearized p2398 (vector containing the APP/neo sequences fused to the mouse APP exon 16 sequences) in a standard ligation reaction and incubated overnight at 14°C. The resulting targeting vector, p2399 (350 μg), was linearized with 320 units of Pmel (cat#560L) in buffer 4 (NEB) and 0.1 mg/ml BSA and incubated overnight at 37 °C. Protein was removed by adding sodium acetate pH 5.2 to 0.3 M and extracting twice with Tris-Cl buffered phenol and extracting once with chloroform and ethanol precipitating at -20 °C for overnight.

Construction of the completed targeting vectors containing the human APP London-FAD. Swedish/London-FAD. and "Swedish-FAD APP713 mutation: These three targeting vectors were constructed by ligating four separate fragments with one of these fragments containing one of the FAD mutations. The seminal targeting vector construct, p2398, was digested in three independent reactions to obtain three of the specific fragments. (1.) Five μg of p2398 were digested with 15 units of Aflll (cat #520S) in buffer 2 with 0.1 mg/ml BSA at 37 °C for overnight. To this reaction was added enough buffer 3 to adjust the concentration to 100 mM NaCl and 30 units of Notl and incubated at 37 °C for 3 hours. (2.) Twenty μg of p2398 were digested with 24 units of Bglll, 20 units of Notl, and 0.1 mg/ml BSA in buffer 3 at 37 °C for overnight. (3.) Twenty μg of p2398 were digested with 20 units of Aflll, 10 units of Clal (cat#197L), and 0.1 mg/ml BSA in buffer 4 at 37°C for overnight. All three digestion reactions were run on 0.8% low melt agarose gels in 0.5X TAE buffer at 70 volts for 3 hours. From digestion reaction (1.) an 8Kb fragment containing the neomycin-murine APP intron 16-pBluescript sequences was excised, from reaction (2.) a 2Kb fragment containing murine APP intron 15-exon 16 sequences was excised, from reaction (3.) a 2Kb fragment containing human cDN A/poly A sequences was excised and all the fragments were isolated away from the agarose using the Qiaex Gel Extraction kit. The last fragments to isolate were the three 700bp human APP FAD containing fragments. Twenty-five μg of each of the ρMTI-2385 London (not shown), ρMTI-2385 Swedish/London (not shown), and pMTI-2385 Swedish-FAD 713 (not shown), vectors were digested with 24 units of Bglll, 15 units of Clal, and 0.1 mg/ml BSA in buffer 4 at 37°C for overnight. The 700 bp bands from these digestions were isolated away from the agarose using the identical protocol as above. A four-part standard ligation reaction was combined using 25 ng of the 8 Kb Aflll/Notl fragment, 250 ng of the 2 Kb Aflll/Clal fragment, 300 ng of the 700 bp Bglll/Clal fragment, and 250 ng of the 2 Kb Notl/Bglll fragment and incubated at 14 °C for 24 hours. One-sixth of the ligation reaction was used to transform E.coli XL-1 blue cells in a standard protocol. The resulting constructs were designated as p2450 (London-FAD), p2451 (Swedish/London-FAD), and p2452 (Swedish-FAD APP713)(not shown). The final step for each individual plasmid was to clone the TK gene fragment with Notl ends into it. Five μg of each plasmid, p2450, p2451, p2452 were digested with 20 units of Notl in buffer 3 at 37°C for 3 hours. To dephosphorylate the vector, 10 units of CIP were added to the digestion reaction and incubated at 37 °C for 1 hour. The phosphatase was heat inactivated at 75 °C for 10 minutes. The linearized DNA band was excised and isolated away from the agarose using the Qiaex DNA Gel Extraction kit and the protocol provided as stated above. Fifty ng of each dephosphorylated vector was ligated to 300 ng of the 3 Kb

Notl TK gene fragment in a standard ligation reaction. The resulting plasmids were designated as p2453 (London-FAD; Figure 6: Figure 19), ρ2454 (Swedish/London-FAD; Figure 7; Figure 20), and p2455 (Swedish- FAD APP713; Figure 8: Figure 21). Each of these three targeting vectors (500 μg) were linearized with 500 units of Ascl in buffer 4 at 37° C for overnight. The DNAs were cleaned away from the enzymes by phenol/chloroform extractions as stated in the section "Cloning of the HSV thimidine kinase (TK) gene into the targeting vector". Linearized plasmids were electroporated into ES cells.

miniSouthern-blot analyses

DNA sample preparation: Potential clones were grown in a 96 well plate format. Samples were lysed with the addition of 50 μl of Lysis Buffer [10 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 10 mM NaCl, 0.5% Sarcosyl, and 1 mg/ml Proteinase K (added fresh)] per well and incubated overnight at 65 °C in a humidified chamber. The DNA is precipitated by the addition of 100 μl of 75 mM NaCl in ethanol followed by incubation at room temperature for 15-30 minutes. The DNA is then washed 3x with 150 μl of 70% ethanol added drop by drop to each well. After the final wash, the plate is inverted and allowed to air-dry for 5 - 10 minutes. While the plate is drying, the Restriction Enzyme Cocktail (lx Restriction Buffer specified for the enzyme being used, 1 mM Spermidine, 100 μg/ml Bovine Serum Albumin, and 10 - 20 units of enzyme) is prepared. 30 μl of this cocktail is then added to each well. Incubate overnight at the restriction enzyme's required temperature in a humidified chamber. The next day add 4-5 ml of loading dye (10 mM Tris-HCl pH 6.0, 0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll (Type 400; Pharmacia) in water, and 30 mM EDTA.) and store at -20°C.

Agarose Gel Electrophoresis: A large gel tray (Owl Scientific) is prepared with three 36-teeth combs (evenly distributed along the length of the tray) and 400 ml of molten agarose (FMC). This size of gel will accommodate one 96-well mini-Southern digest plate. The samples were electrophoresed for approximately three hours at 120 V. After the electrophoresis was complete, the gel was denatured in 0.25 M HC1 (2 x 7 minutes at room temperature) and then equilibrated in 0.4 N NaOH (1 x 20 minutes at room temperature). An overnight alkaline capillary transfer is set up in 0.4 N NaOH with Gene Screen Plus (DuPont NEN). The next day the membrane was neutralized in 2x SSC for 5-10 minutes and then UV cross- linked (Stratagene). The membrane is then stored dry until hybridization. Prehybridization was carried out in 1 M NaCl (Gibco BRL), 10% Dextran Sulphate (Pharmacia), 1 % SDS (Gibco BRL), and 200 μg/ml salmon sperm DNA(Stratagene) for at least one hour at 65° C in a Robbins Hybridization

Oven. The probe of interest was then labeled according to the standard protocol contained in the Prime-It II random prime kit (Stratagene). The specific activity of the probe was approximately 1 x 109 dpm/μg. It was then added directly to the prehybridization mixture at a concentration ^"1 x 106 dpm/ml. The filter (s) were then hybridized for 16 hours at 65 °C in the hybridization oven. The initial post - hybridization wash was carried out for 5-10 minutes at room temperature in 2x SSC (3 M NaCl, 0.3 M Sodium Citrate Dihydrate), 1 % SDS. A stringent wash was then performed in lx SSC, 0.1 % SDS at 65 °C for 30 minutes. The filter was then placed into a seal-a-meal bag and placed into a Fuji Phosphoimager for interpretation.

Confirmatory Southern Blots

Preparation of High Molecular Weight DNA from Cells: In order to confirm targeted clones identified in the mini - Southern paradigm, cell pellets expanded from these clones are analyzed for accurate recombination events at both the 5' and 3' ends of the targeting vector. 1 ml of Cell Lysis

Buffer (100 mM NaCl, 50 mM Tris pH 7.5, 10 mM EDTA pH 8.0, and 0.5% SDS) and 20 ml of freshly prepared 40 mg/ml Proteinase K (Boehringer-Mannheim) was added to each cell pellet. The tubes were rocked overnight at 65 °C. The next day, an equal volume amount of isopropanol was added and the tube inverted several times to precipitate the DNA. The DNA was then spooled onto a flame sealed micropipette and rinsed once in 70% ethanol, once in 100% ethanol, and then air dried. The pipette was broken off into a sterile Eppendorf tube and the DNA dissolved in

200 μl of sterile TE overnight at room temperature. The DNA is then stored at 4°C until restriction enzyme analysis.

Agarose Gel Electrophoresis: Restriction enzyme digested DNA is gel analyzed as described above in the mini-Southern methods except the number and sizes of the combs vary. Denaturation, renaturation, and capillary transfer were performed as described previously. Probes of interest were also labeled in the same manner as described above. Interpretation of results were facilitated by phosphoimaging as previously described.

Gene-tørgeting in ES cells

Culture of ES cells: Procedures were performed essentially as describe in E. J. Roberstion (Robertson, 1987) . ES cell were propagated using Mitomycin C treated SNL76/7 STO feeder cells (cell line obtained from A. Bradley) and modified DMEM culture media (supplemented with 15%

FCS,1X GPS,1X BME).

Electroporation of ES cells: DNA was linearized with the appropriate restriction enzyme then extracted with an equal volume of phenol/chloroform and once with an equal volume of chloroform and precipitated with 2.4 volumes of ethanol. The DNA was resuspended at 1 mg/ml in sterile 0. IX

TE (25 ml of DNA per electroporation). Embryonic stem cells (80% confluent) were passaged 1:2 the day before electroporation. Cells to be electroporated were fed 4 hours before harvesting. The cells were trypsinized and resuspend in media (cells from 2 x 10 cm plates can be combined in a total volume of 10 ml in a 15 ml tube). The cells were pelleted and resuspend in 10 ml PBS at a density of 11 x 10⁶ cells/ml. The appropriate amounts of DNA and cells were mixed together in a 15 ml tube (25 ml of DNA and 0.9 ml of cells for each electroporation) and allowed to sit at room temperature for 5 minutes. The cell/DNA mixture (0.9 ml) was transferred to electroporation cuvettes and an electrical current was passed through the solution (using Biorad GenePulser at 230V and 500 mF). The cells were then transferred to culture plates with feeder cells (up to 2 x 10⁷ cells/ 100 mm plate or 6 x 10⁶ cells/60 mm plate). After 24 hours of culture in modified DMEM the cells were cultured in DMEM selection containing G418 and 0.2 mM FIAU. Resistant colonies may be picked as early as 8 days, are best around 10-11 days, but may be recovered up to 18-21 days after the electroporation. Picked colonies are transferred to 96 well plates with feeders cells and screened for gene-targeting events by mini-Southtern-blot analysis (see below).

Production of chimeric mice: Procedures were performed essentially as described by A. Bradley (Bradley, 1987) . Host 3.5 day blastocysts were derived from timed matings of C57BL/6 mice and cultured in M16 media.

Approximately 14 targeted ES cells were injected into each blastomere. Surviving blastocysts were then surgically reimplanted (approximately 12 per animal) into pseudopregnant ICR female mice essentially as described (A. Bradley). Chimeric mice were born about 17 days after implantation.

Genotype analyses of transgenic mice

Identification of mice possessing the targeted human APP cDNA by PCR screening: When mice were older than 2 weeks of age their tails were biopsies to obtain genomic DNA for analysis. One centimeter pieces of tail were prepared using the QIAamp Tissue Kit (Qiagen cat# 29304) and following the protocol provided. Genomic DNA was eluted in 150 μl of 10 mM Tris-Cl pH 9 and used in two independent PCR assays; (1) to determine the endogenous mouse APP allele that remained intact: total reaction volume of 50 μl - 5 μl of genomic tail DNA (approximately 1 μg), 5 μl of 10X buffer 8 (Stratagene cat#200430), 5 μl of 2 mM dNTP mix, 200 ng of oligonucleotide KC 125 (5 ^* ACTTTGTGTTTGACGC3 ' ) , 200 ng of oligonucleotide KC132 (5'CAGTTTTTGATGGCGG3'), 1 unit of Perfect Match Polymerase Enhancer, 2.5 units of AmpliTaq and 100 ng each of oligonucleotides 6&7 and (2) to determine the targeted mouse APP allele: total reaction volume of 50 μl - 5 μl of genomic tail DNA (approximately 1 μg), 5 μl of 10X buffer 8 (Stratagene cat#200430), 5 μl of 2 mM dNTP mix,

200 ng of oligonucleotide KC125 (5ΑCTTTGTGTTTGACGC3'), 200 ng of oligonucleotide KC 131 (5^*GATGATGAACTTCATATCCTG3'), 1 unit of Perfect Match Polymerase Enhancer, 2.5 units of AmpliTaq and 100 ng each of oligonucleotides 6&7. The reactions were run in a Perkin Elmer turbo 2400 thermal cycler. The parameters of the run were as follows: one cycle at 94°C for one minute, 30 cycles at 94°C for 30 seconds-56°C for 50 seconds-70°C for two minutes, maintain at 10°C indefinitely.

Oligonucleotides 6

(5'CCTCGGCCTTTGGTGTGTGTTTTATGACATGACCCCCTTGA) & 7 (5 ' C ACCCTGTTGTC AATGCCTCTGGGTTTCCGCC AGTTTCG3 ^* ) are homologous to mouse ribosomal protein L32 sequences within intron 2 and exon 3, respectively, and used as an internal DNA control signal. One-fifth of each PCR reaction was run on a 6% polyacrylamide gel (Novex cat#EC6265) in IX TBE (89 mM Tris borate, 2 mM EDTA) buffer at 125 volts for 35 minutes and stained in 1 mg/ml EtBr for 15 minutes and photographed.

RNA analyses

RNA isolation: Total brains were dissected and flash frozen on dry ice from two negative litter mates, two heterozygous targeted mice, and two homozygous targeted mice. In addition, kidneys and tails were also removed from these mice and flash frozen. The brains were divided in half, one for the RNA analysis and the other for protein analysis. To one-half of each brain was added 5 ml RNAzolB (Tel-Test, Inc. cat#CS-105) and the tissues were homogenized using a Brinkman Polytron at medium speed for 20 seconds. Chloroform, 500 μl, was added to the homogenized tissue and shaken well for 10 seconds and incubated on ice for 15 minutes. The samples were spun in a tabletop Sorvall centrifuge at 1500Xg for 20 minutes at 4°C.

The aqueous phase was removed and added to an equal volume of isopropanol, mixed, and incubated on ice for 15 minutes. The samples were spun in a Sorvall RC-5B centrifuge with an SS-34 rotor at 7500Xg at 4°C for 25 minutes. The supernatants were removed and the pellets were rinsed twice in cold 70% EtOH and air dried. The total RNA pellets were resuspended in 500 ml H₂0 and incubated at 65 °C for 10 minutes to more easily get the RNA into suspension. These RNA samples were used to obtain polyadenylation specific mRNA using the PolyAtract mRNA isolation system

III kit (Promega Z5300). The protocol followed was provided by the supplier and yields ranged from 3 to 6 μg of mRNA.

Northern blot analyses: These samples were then used in a Northern blot to see the sizes of these targeted hybrid APP transcripts. The RNA was run on a 1.2% agarose (FMC cat# 50072) , 2.2 M formaldehyde gel prepared as follows: 0.6g agarose in 36 ml H₂0 were melted in a microwave and placed at 60°C. When the gel cooled to 60°C , 5 ml of 10X MOPS (0.4 M MOPS (Sigma MESA M-5755) pH7, 0.1 M sodium acetate, 10 mM EDTA pH8) running buffer and 9 ml of 37% formaldehyde (pH >4) were added, mixed and left at 45 °C until ready to pour. The RNA samples were prepared in a total volume of 30 μl - 3 μl 10X MOPS buffer, 5.25 μl 37% formaldehyde, 15 μl formamide, and 6.75 μl mRNA (0.5 μg) were mixed well and incubate at 55 °C for 15 minutes. To this was added 6 ml formaldehyde loading buffer (1 mM EDTA ρH8, 0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol) and 1 ml 1 mg/ml EtBr. The samples were loaded into the gel and run at 5V/cm (55-75 V) for 3hr in IX MOPS buffer. The gel was rinsed in H₂0 several times and soaked in 0.05N NaOH for 30 minutes under gentle shaking. The gel was then equilibrated twice for 15 minutes in 20X SSC and transferred by wick assembly for 16 hours in 20X SSC. The membrane used for transferring was Hybond-N-I- (Amersham cat#RPN2020B) which is a 0.45 micron nylon membrane. After transfer was completed the membrane was rinsed in 2X SSC for 10 minutes and UV cross- linked in a Stratalinker mentioned earlier. The membrane was pre-hybridized in 10 ml 0.5 M sodium phosphate pH7, 1 % BSA, and 7% SDS for 4 hours at 65 °C in a Robbins Hybridization Oven (model 400). This solution was removed and replaced with fresh hybridization solution and 6x107 counts of denatured APP probe and 8x105 counts of denatured mouse beta-actin probe and hybridized overnight at 65 °C. The membrane was washed in 2X SSPE,

0.1 % SDS at 25 °C for 10 minutes, twice, and then washed in IX SSPE, 0.1 % SDS (pre- warmed) at 65 °C for 15 minutes. The membrane was exposed to a phosphoimaging screen for 24 hours and developed.

Probes for Northern blot: Both the APP probe (homologous to the murine and human sequences) and the murine beta-actin probe were prepared in identical protocols. The APP DNA used to make the probe was an Nrul/Xhol 900bp fragment from p2385B. The murine beta-actin 430bp DNA used for the probe came from a PCR reaction where the exon 3 of B-actin was amplified using these two oligonucleotides: KC137 (5'GTTTGAGACCTTCAACACCC3') and KC138

(5'GAAGGAAGGCTGGAAAAGAGCC3'). The probes were labeled using the Prime It II kit (Stratagene cat#300385) and following the protocol provided. After the reactions were stopped they were put over a G-50 spin column (5 '-3' cat# 5303-633329) to remove the un-incorporated nucleotides. To increase the level of APP-specific mRNA from the polyA selected

RNA, the samples were annealed to an APP specific oligonucleotide (RA49- 5'CGATGGGTAGTGAAGCA3')) that was homologous to both the murine and human sequences approximately 40nt 3' of the stop codon. The assay was performed using the Superscript II RT-PCR kit (Gibco/BRL cat# 18089- 011). In a reaction volume of 14 μl was combined 0.1-0.15 μg poly A mRNA and 600 ng RA49 and incubated at 70°C for 10 minutes and 4°C for 10 minutes. To this was added 2 μl 10X synthesis buffer, 1 μl 10X dNTP mix, 2 μl 0.1 M DTT, and 200 units of Superscript II reverse transcriptase (all supplied by the kit) and the incubations continued at 25 °C for 10 minutes, 42°C for 50 minutes, 70°C for 15 minutes, and 4°C for 10 minutes. The reactions were then treated with 2 units of RNaseH for 20 minutes at 37 °C and then placed on ice. After the RNA was removed from the cDNA the next step was the amplification reaction: 20 μl of cDNA reaction mix, 8 μl

10X synthesis buffer, 300 ng of oligonucleotide KC56 (5'GTGAAGATGGATGCAGAATTC3'), 300 ng of oligonucleotide KC56 Swedish (5'GTGAATCTAGATGCAGAATTC3'), 600 ng of oligonucleotide RA49, and 5 units of AmpliTaq in a total volume of 100 ml. The amplification was run in the Perkin Elmer turbo 2400 using the same parameters as stated in "Identification of mice possessing the targeted human APP cDNA by PCR screening". The RT-PCR reactions were subjected to restriction enzyme digestions taking advantage of the restriction site polymoφhism between the murine and human APP sequences. One-tenth of the RT-PCR reaction was digested with 30 units of Sail and 0.1 mg/ml BSA in its ideal buffer at 37 °C for 2 hours, another set was digested with 30 units of Styl in buffer 3 at 37° C for 2 hours. The digests were run out on a 4% polyacrylamide gel in IX TBE at 150 volts for 1 hour and stained in 1 mg/ml EtBr for 15 minutes and photographed. All oligos were provided by Midland and all restriction enzymes by NEB.

Protein Analysis

Tissue Extraction: This protocol is generally used for mouse tissue with no more than several hundred mgs of tissue available, therefore all volumes must be kept to a minimum. Tissue was homogenized in 1 ml of RAB buffer (0.1 M MES pH 7.0, 0.75 M NaCl, 0.5 M MgCl₂, 1 mM

EGTA, 1 mM DTT) containing proteinase inhibitors. The protease inhibitor cocktail contains lx Aprotonin (0.41 trypsin inhibitor units/mg protein), lx PMSF (2 mM in isopropanol), lx Protease inhibitor mix (chymostatin, leupeptin, antipain, and pepstatin) each at 50 μg/ml in DMSO, and 1 mM EDTA. A 7 ml dounce tissue grinder (Wheaton) was used for homogenization. The tissue homogenate was spun at 40K in the Beckman TL100 using the fixed angle rotor for one hour. The supernatant from this spin was saved as it contains the soluble APP. The pellet was homogenized in 1 ml of RAB plus protease inhibitors and 30% sucrose (Sigma). Spin for one hour at 40K in the Beckman TL100. This serves as a wash and demylelinating step. Discard the supernatant from this spin and homogenize the pellet in 1 ml of RIP A buffer (150 mM NaCl, 1 % NP-40, 0.5% deoxycholate (Na salt), 0.1 % SDS, and 50 mM Tris-Cl pH 8.0). This should contain the membrane associated form of APP. The amount of protein can then be quantitated by using the BCA Protein Assay Reagent Kit (Pierce). This quantitation allows equal amounts of total protein to be loaded on polyacrylamide gels and direct comparisons of transgenic and non-transgenic expression patterns and levels to be made.

Immunoprecipation: The final adjusted volume of the immunoprecipitation was 1 ml in RIP A buffer. The amounts of antigen and antibody to add varied from experiment to experiment depending on the concentrations of both. Antibody and antigen were incubated for two hours at 4°C while gently spinning on a rotating wheel. 50 μl of goat anti-mouse or anti-rabbit IgG bound to agarose (Sigma) was added to the antigen/antibody and incubated for another two hours at 4°C on the rotating wheel. Agarose IgG-antigen/antibody complex was rinsed by pelleting at 12,000 x g for 1 min. and then removing the supernatant. Then 500 μl of ice cold RIPA buffer was added to the pellet, resuspended, and incubated for 10 minutes on ice. The samples were then spun at 4°C. The rinses were repeated twice more, but the 10 minute incubation step was omitted. To the rinsed pellet, was added 50 μl of lx sample buffer (Novex)plus 2 ml of beta- mercaptoethanol (Aldrich). Samples were boiled for 10 minutes and spun for 1 minute at room temperature. The supernatant was transfered to a fresh tube and store at -20°C.

Western Blotting: Polyacrylamide gel electrophoresis (PAGE) and electroblottting were accomplished utilizing the X-Cell II Gel and Blot

Module (Novex) and pre-casted polyacrylamide gels (Novex). The selection of a particular separation scheme depended on what form of the Alzheimer Precursor Protein (APP) was being examined. For C-terminal fragments 16% Tris-Tricine gels were utilized, holo APP utilized 10-20% Tris-Tricine gels, and to elucidate form differences (Kunitz vs. 695) of the holo- APP, 6%

Tris-Glycine gels were used. Samples prepared as described above were loaded onto gels and electrophoresed at 120V for approximately 90 minutes. The gels were then transferred to nitrocellulose membranes (Novex) for 1-2 hours at 30V. Non-specific sites were then blocked by incubation of the filter in 5% non-fat dry milk (NFDM) for 1 hour at room temperature while gently rocking. Primary antibody was then added at a dilution of 1 :500 in 5-10 ml of NFDM, added to the membrane and sealed in a seal-a-meal bag. This was incubated overnight at room temperature while gently rocking. The membrane was then rinsed for 1 hour at room temperature with several changes of 5% NFDM. A ³⁵S labeled secondary antibody (Amersham), either anti-mouse IgG or anti-goat IgG, was then added and incubated for 1 hour at room temperature while gently shaking. The membrane was then rinsed for 15-30 minutes in 5% NFDM and then equilibrated into lx phosphate buffered saline (PBS, Gibco BRL) for 15 minutes. The filter was then dried and either placed on a phosphoimaging plate or with a piece of

X-OMAT X-ray film (Kodak). APP Antibodies: Monoclonal antibody (MAb) 4G8 (Senetek) was used for the immunoprecipitation of APP holoprotein and C-terminal fragments at a dilution of 1:100 (^"10-20 μg/ml). MAb 286.8 (BRC) was used for the immunoprecipitation of APP holoprotein at a dilution of 1 : 100 ("10-20 μg/ml). MAb 6E 10 (Senetek) was used as a detection reagent on

Western blots at dilutions of 1:500. Polyclonal antibody (PAb) 369 (generously provided by Dr. Sam Gandy) was used for both the immunoprecipitation of APP holo-protein (1 : 100) and for a detection reagent for C-terminal fragments (1:500). MAb 22C11 (generously provided by Dr. Konrad Beyreuther) was used as a detection agent for APP holo-protein at a dilution of 1:500.

FAD-m/hAPP gene products expressed in transgenic mouse lines

Transgenic mouse lines ES5007, ES5103, ES5401, and ES5403 were generated by mutating the mouse APP gene via homologous recombination in embryonic stem (ES) cells (see below). The gene products expressed in the transgenic mouse lines are described schematically in Figure 10. m/hAPP770 represents the largest (770 amino acid residues) of the various alternative splice forms of protein expressed by each mutated mouse APP gene. m/hAPP exhibits amino acid sequence identity with mouse APP with the exception of those residues indicated by (asterisks, *). In all cases the beta- amyloid (bA4) domain (Asp672 to Thr714; 43 amino acid residues) has been "humanized" by the introduction of three amino acid substitutions (as indicated by green asterisks); Gly(676) to Arg, Phe(681) to Thr, and Arg(684) to His. Transgenic mouse line ES5007 also has the Swedish-FAD mutation [Lys,Met(670,671) to Asn,Leu] introduced into the mouse gene.

Transgenic mouse lines ES5401 and ES5403 have the London-FAD mutation [Val(717) to Ilu] and transgenic line ES5103 carries both Swedish and London FAD mutations. In addition to the Swedish FAD and "human" mutations, a transgenic mouse line ES5215 can also be produced which has a premature stop codon introduced at position 714.

Gene-Targeting Vectors

The targeting vectors were designed in such a way as to facilitate the integration of human cDNA sequences into mouse exon 16. The targeting constructs function as replacement-type vectors with both positive (neomycin resistance gene) and negative (HSV TK gene) selection genes (figure 11). To facilitate homologous recombination, a mouse genomic clone encompassing exon 16 was obtained by screening a mouse genomic lambda library. A lambda clone, "35A", was identified which contained an intact exon 16

(figure 1). Nco I fragments of mouse genomic clone 35 A were subcloned into the BSII SK+ vector and the subclones (pRA3, pMTI-2396, and pN2C4) were characterized by DNA sequence and restriction enzyme analyses (see Figures 2, 3, and 4). The 5.5 Kb Nco I DNA fragment (subcloned into pMTI-2396) contains APP exon 16 and "1.9 Kb and ^"3.5 Kb from introns 15 and 16 respectively (Figure 2). The Nco I DNA fragment, containing exon 16, was the template upon which the gene-targeting vectors were constructed.

The gene-targeting vectors were designed so that mouse exon 16 gene sequences were fused (at a common Bgl II site) with human cDNA sequences which encode the remainder of exon 16 and exons 17 and 18 (figure 11). The mouse and human cDNA sequences encode the identical protein sequence with the exception of 3 amino acid differences (shown as green asterisks) which reside within the beta-amyloid domain. The mouse genomic-human cDNA fusion effectively "humanized" the beta-amyloid domain and facilitated the introduction of specific FAD mutations while leaving the remainder of mouse APP protein sequences unchanged (see Fig 13). The human cDNA was mutagenized to encode either the " Swedish "- FAD, "London"-FAD , "Swedish"/"London"-FAD (shown here), or "Swedish" -FAD APP713 mutations (shown as black asterisks) of APP (see Fig. 10 and Table I). The mutagenesis of the "Swedish"-FAD mutation also incoφorated a new Xba I restriction site. Proper RNA processing was ensured by fusing the 3 '-end of the human cDNA sequence with human genomic sequences which contain transcription termination and polyadenylation signals from the human APP gene. A neomycin gene was inserted in-between the 3 '-end of the human APP polyadenylation signal and mouse APP intron 16 sequences. Targeting vector pMTI-2398 (Swedish-

FAD) contained the neomycin resistance gene and not the HSV TK gene. This vector was linearized with Pme I and was used to generate transgenic mouse line ES5007. (Tables I and II).

For the remaining three targeting vectors, a HSV Tk gene was inserted into the clone in such a way that its placement was outside of the genomic domains homologous to mouse (Figure 11 ; as shown or in the opposite orientation; the orientation was not critical). Targeting vector pMTI-5453 encodes London-FAD m/hAPP, targeting vector pMTI5454 encodes Swedish/London-FAD m/hAPP, and targeting vector pMTI-5455 encodes Swedish-FAD m/hAPP713. These targeting vectors were linearized with Asc

I and were used to generate transgenic lines. (Tables I and II).

Gene-targeting in embryonic stem (ES^ cells

The targeting vectors were designed to function as replacement-type vectors with both positive (neomycin resistance gene) and negative (HSV TK gene) selection genes. After electroporation of the targeting vector into ES cells, G418 drug treatment selected for ES cells which had integrated the targeting vector (including the neomycin resistance gene) into the mouse genome. The majority of G418 resistant ES cell clones had targeting vector integrated at random locations of the genome. These ES cell clones retained an intact HSV TK gene and were not desired. The clones containing random integrations could be eliminated by treatment with FIAU selection media which is toxic only to cells expressing HSV TK. If, as desired, the mouse APP gene is targeted via a double-crossover homologous recombination event, the flanking non-homologous HSV TK DNA sequences are lost (as shown in fig 12) and the ES cells are resistant to FIAU treatment.

Homologous recombination between mouse APP exon 16 locus and the gene-targeting vector fundamentally alters the manner by which the gene encodes APP (see figure 13). Normally, the beta-amyloid, transmembrane, and cytoplasmic domains of mouse APP are encoded by three separate exons. In addition, the coding region for the beta-amyloid domain resides both on exons 16 and 17. After homologous recombination with the gene targeting vector, however, mouse exon 16 gene sequences are fused with human cDNA sequences. Mouse exons 17 and 18 are now displaced down-stream from the neomycin resistance gene and are inactive. The human cDNA now functions in place of mouse exons 16, 17, and 18 to encode APP. Therefore, the beta- amyloid, transmembrane, and cytoplasmic domains of mouse APP are now encoded by human cDNA sequences. The gene products of this new mouse genomic-human cDNA fusion are designated m/hAPP. Human cDNA sequences (exons 16, 17, and 18) encode the identical protein sequence with the exception of 3 amino acid differences (shown as green asterisks) which reside within the beta-amyloid domain. The mouse genomic-human cDNA fusion effectively "humanizes" the beta-amyloid domain and facilitates the introduction of specific FAD mutations (shown as black asterisks) while leaving the remainder of mouse APP protein sequences unchanged. The human cDNA has been mutagenized to encode either the "Swedish "-FAD, "London"-FAD , "Swedish'VLondon"- FAD (shown in figure 2c), or "Swedish"-FAD APP713 mutations of APP (see also Fig. 10 and Table I).

Identification of targeted ES cell clones

After electroporation of each targeting vector (see Table I), ES cells were cultured for approximately 2 weeks in the presence of both positive

(G418) and negative (FIAU) selection compounds. Four hundred G418/FIAU resistant ES cell colonies (clones) were then individually picked and cultured separately in 96 well culture dishes. The culture dishes were replica-plated, one set of copies was frozen to maintain the clones and the other replicate set was utilized for genetic analyses. From each well, DNA was extracted, digested with restriction enzyme, and analyzed by miniSouthern-blot analyses (see below). ES cell clones which appear to contain a targeted APP gene locus were thawed and expanded in culture. Gene-targeting was confirmed by Southern-blot analyses using DNA extracted from these expanded clones prior to introduction of the ES cell into the mouse germline (see below).

The mutagenesis of human cDNA's to encode the Swedish-FAD mutation also created a new Xba I (shown as X) restriction enzyme site (Figure 14). Incoφoration of human FAD cDNA (shown in red) into the targeted m/hAPP gene locus thus changes the pattern of DNA fragments generated after digestion of this locus with Xba I. Using Southern-blot analyses, ES cell clones having the targeted m/hAPP gene can be distinguished from neomycin resistant ES cell clones having undesired random integrations of the targeting vector. The mouse exon 16 gene locus can be detected using a 3Kb Nco I (N) DNA fragment from intron 15 of the mouse APP gene as probe. Digestion of the mouse APP gene with Xba I generates an approximately 9 Kb DNA fragment whereas Xba I digestion of the targeted Swedish-FAD m/hAPP gene gives an approximately 5 Kb DNA fragment when detected by Southern-blot analysis (see figure 14). This detection strategy applies to the Swedish-FAD m/hAPP, Swedish/London- FAD m/hAPP, and Swedish-FAD APP713 mutations.

Mini-Southern blot analysis identified 4 ES cell clones which appeared to contain the targeted Swedish-FAD m/h APP locus (data not shown).

These clones were expanded and subsequent Southern-blot analysis demonstrated that ES cell clones A79, A80, and B12 contain the Swedish FAD APP mutation while clone A72 did not (Figure 15). DNA extracted from ES cell pellets was examined by Southern-blot analysis using the restriction enzyme Xba I as described in Figure 14. A single ^~9Kb DNA fragment is observed in non-targeted ES cells whereas targeted ES cell clones exhibit both the non-targeted allele (^"9Kb fragment) and the FAD mutant allele giving rise to a ^"5 Kb band. Transgenic mouse line ES5007 was derived from ES cell clone B12 (Table I). The remaining positive ES cell clones failed to establish germline transmission of the FAD mutation.

Initial miniSouthern-blot analyses identified five ES cell clones which appeared to contain Swedish/London FAD APP double mutation (data not shown). DNA extracted from pellets of expanded ES cell clones was examined by Southern-blot analysis using the restriction enzyme Xba I as described in Figure 14. This analysis confirmed that ES cell clones C82,

C87, D25 and D92 contained the Swedish/London FAD m/hAPP double mutation while clones C52 and D49 did not. Transgenic mouse line ES5103 was derived from ES cell clone C87 (see Table I). The remaining positive ES cell clones failed to establish germline transmission of the FAD mutation. Confirmatory Southern-blot analyses identified multiple clones which carry the Swedish-FAD m/hAPP713 mutations (data not shown).

While identical in all other respects, the targeting vector encoding London-FAD m/hAPP does not carry the Xba I restriction site associated with the Swedish mutation. It was necessary, therefore, to identify restriction enzymes which could distinguish between un-altered ES clones and those ES cell clones containing a targeted m/hAPP allele. The restriction enzymes Bel I and BpM I were found to distinguish between DNA from a non-targeted ES cell clone (clone Al) and DNA from clone A21 which contains the Swedish- FAD m/hAPP gene locus (data not shown, see also figure 19). Bel I and

BpM I can be used to identify targeted clones derived from any of the aforementioned gene-targeting vectors.

Using restriction enzyme Bel I, miniSouthern-blot analysis identified 6 ES cell clones which appeared to contain the London-FAD mutation (data not shown). Confirmatory Southern-blot analysis , using restriction enzyme BpM

I, demonstrated that ES cell clones D12, D60, D70, D74, and D90 contained the London-FAD m/hAPP targeted locus while clone D45 did not (figure 18). After digestion with BpM I, three DNA fragments (^"6 Kb, ^"3.8 Kb, and ^"2.2 Kb) are observed in non-targeted ES cells whereas targeted ES cell clones exhibited an additional ^"4.8 Kb DNA fragment from the targeted allele (see

A21 targeted for Swedish mutation. Transgenic mouse lines ES5401 and ES5403 were derived from ES cell clones D12 and D60 respectively (Table I). The remaining positive ES cell clones failed to establish germline transmission of the FAD mutation.

Germline-transmission of targeted m/hAPP genes

ES cells, confirmed to contain a targeted m/hAPP allele, were injected into the blastocoel cavity of a 3.5 day pre-implantation embryo (blastocyst). The injected blastocysts were then surgically reimplanted into pseudopregnant fosters and chimeras were born after approximately 17 days. The ES cells were derived from the 129SVEV inbred mouse strain which has a dominant agouti coat color gene. The blastocysts were derived from the C57BL/6 inbred mouse strain which carries a recessive black coat color gene. The coat color of chimeric mice whose cells are predominately derived from the ES cells (designated as "high percentage chimeras") is mostly agouti with small patches of black. To establish germline transmission of the targeted APP gene, high percentage chimeric male mice were mated with either 129/SVEV inbreed or black Swiss outbreed females. The genotype of offspring from ES5007, ES5103, ES5401 and ES5403 breeding pairs was determined by either Southern-blot or PCR analyses.

The Southtern-blot analyses could distinguish between non-targeted, heterozygous, and homozygous progeny mice. The analyses utilized either Bel I or BpM I restriction enzyme, and the ^"3.0Kb intron 15 DNA fragment as probe (see Figure 14 for description of probe). A Southern-blot characterizing DNA from progeny of heterozygous ES5007 breeding pairs was performed. The technique can be applied to all the aforementioned transgenic lines. Bel I digestion of non-transgenic (wt) mouse DNA and non- targeted ES cell DNA generated ^"16 and ^"8.5 Kb DNA fragments. However, Bel I digestion of heterozygous transgenic mouse DNA and targeted ES cell DNA generated ^"16 , ^"8.5, and ^"8.0 Kb DNA fragments. Digestion of homozygous mouse DNA with Bel I liberated ^"8.5 and ^"8.0 Kb DNA fragments. BpM I digestion of non-transgenic (wt) mouse DNA and non-targeted ES cell DNA generated ^"6.0, ^"3.8, and "2.2 Kb DNA fragments. Bpm I digestion of heterozygous transgenic mouse DNA and targeted ES cell DNA generated ^"6.0 , ^"4.8, ^"3.8, and ^"2.2 Kb DNA fragments Digestion of homozygous mouse DNA with BpM I liberated "6.0 , ^"4.8, and ^~2.2Kb DNA fragments.

The genotype of offspring from ES5007, ES5103, ES5401 and ES5403 breeding pairs was also determined by PCR analyses using a combination of oligo pairs specific to human APP (H) and mouse APP (M) sequences. Like the Southern-blot technique, PCR analysis can distinguish between non- targeted, heterozygous, and homozygous progeny mice. As an internal standard, a 154 bp region of the mouse ribosomal subunit L32 gene is amplified using the PCR oligo pair 6 and 7 (Figure 9). This control reaction was performed in each reaction. A 118 bp region specific to the mouse APP gene is amplified using the "M" oligo pair (oligos KC125 and KC132; Figure 9) and a 109 bp region specific to targeted m/hAPP gene in amplified using the "H" oligo pair (oligos KC125 and KC131; Figure 9). A PCR reaction using non-transgenic mouse DNA (wt) gives rise to a 118 bp fragment using the "M" oligo pair but no reaction product using the "H" oligo pair. Conversely, a PCR reaction using DNA from transgenic mice homozygous (homoz.) for the targeted m/hAPP gene gives rise to a 109 bp fragment using the "H" oligo pair but no reaction product is observed using the "M" oligo pair. A PCR reaction using DNA from heterozygous transgenic mice (heter.) gives rise to both mouse and human PCR reaction products.

Messenger RNA (mRNA) expression in transgenic mouse brain

Analysis of APP mRNA composition in control mouse and ES5007 mouse brain has been determined using both Northern-blot and rtPCR analyses. RNA analyses have yet to be performed on the ES5103, ES5401, and ES5403 lines (Table II).

APP mRNA transcripts from control and ES5007 mouse brain were detected by Northern-blot analysis using an approximately 900 bp Nru I- Xho I fragment from pMTI-2385B (human APP cDNA) as probe. Mouse beta- actin mRNA was detected using a 430 bp mouse beta-actin cDNA probe (430 bp PCR product generated using oligos KC137 and KC138; see Figure 9) and served as an internal standard. mRNA from human brain (Clonetech) served as a positive control. mRNA from the Swedish-FAD m/hAPP gene was abundantly expressed in the brain from homozygous ES5007 mice. The amount of Swedish-FAD m/hAPP mRNA in ES5007 brain was determined by phosphoimage analysis and shown to be approximately 55 % of the mAPP mRNA levels observed in control mouse brain. In concordance, the APP mRNA levels in heterozygous ES5007 mouse brain were found be approximately 75 % of the level observed in control mouse brain.

The reverse transcriptase-PCR (rtPCR) technique was used to identify mouse APP and m/hAPP transcripts in mouse brain. Homozygous ES5007 mice were found to express mRNA exclusively from the targeted Swedish- FAD m/hAPP gene. No mRNA species containing sequences from mouse APP exons 16, 17, or 18 was detected in homozygotes. Heterozygous ES5007 mice were found to express mRNA transcripts from both mouse APP and Swedish-FAD m/hAPP alleles. mRNA was purified from control and transgenic mouse brain and cDNAs were prepared using reverse transcriptase and oligonucleotide RA49 as primer. A 367 bp DNA fragment was amplified from mouse APP and m/hAPP cDNA by PCR using oligonucleotides KC56 and RA49 (Figure 9). Oligonucleotides KC56 and RA49 exhibit sequence identity with both mouse and human sequences. The mouse and human sequences were distinguished from each other by the presence of a Sty I restriction site in the human cDNA and the absence of the Sty I site in the mouse cDNA. Digestion of the 367 bp PCR product from m/hAPP cDNA generates two fragments (151 bp and 216 bp) while the PCR product from the mouse APP cDNA is not digested and remains unchanged at 367 bp.

APP mRNA from control mouse brain was amplified by rtPCR to generate a 367 bp DNA fragment that was resistant to Sty I digestion. rtPCR amplification of m/hAPP mRNA from the brain of homozygous ES5007 mice gene generated two fragments (151 bp and 216 bp) upon digestion by Sty I.

No 367 bp DNA fragment remained, demonstrating that mouse APP cDNA was not present. All three DNA fragments (151 bp, 216 bp, and 367 bp) were observed after Sty I digestion of rtPCR product derived from heterozygous ES5007 brain transcripts. As a control, Sty I digestion of PCR products from human APP cDNAs derived from human mRNA and mRNA from a HEK293 cell line expressing human APP generated the 151 and 216 bp DNA fragments. As expected, Sty I failed to digest the 367 bp PCR product derived from mouse brain mRNA.

m/hAPP protein expression in transgenic mouse brain

Swedish-FAD m/hAPP protein is expressed in the brain of ES5007 mice. MAb 286.8 specifically immunoprecipitates human APP and but not mouse APP. The epitope for MAb 286.8 has been determined to lie within the N-terminus of the human beta-amyloid domain (P. Graham et al. 1994, Pharma Report MRC 00116). The m/hAPP gene product could be specifically immunoprecipitated from a ES5007 brain homogenate using the monoclonal antibody (MAb) 286.8. APP moieties were then visualized by Western-blot analysis using MAb 22C11 as the detection antibody. MAb 22C1 lean detect both mouse APP and m/hAPP. Therefore, if mouse APP was present after the immunoprecipitation it would have been detected by

MAb 22C11. MAb 286.8 immunoprecipitated baculovirus derived human APP but did not recognize mouse APP in mouse brain homogenates.

The immunoprecipitations were performed using equal amounts of control mouse and ES5007 brain homogenates directly applied to the Western-blot. The relative intensities of the mouse APP and m/hAPP bands were equivalent.

Baculovirus derived human APP was directly applied to the Western- blot. An equal amount of human APP was detected after immunoprecipitation by MAb 286.8. It can be concluded, therefore, that MAb286.8 efficiently immunoprecipitated human APP.

The expression of Swedish-FAD m/hAPP protein was further demonstrated by Western-blot analyses using additional detection antibodies. m/hAPP was immunoprecipitated from a homogenate of ES5007 brain using human-specific MAb286.8. APP was then detected by Western-blot analysis using either the polyclonal antibody (PAb) 369 or MAb 6E10 for detection. MAb 6E10 is human APP specific and recognizes the human beta-amyloid domain. Again MAb 286.8 immunoprecipitates human APP, Swedish-FAD m/hAPP but does not immunoprecipitate mouse APP.

Swedish-FAD m/hAPP protein is expressed in the brain of homozygous ES5007 mice at approximately 87 % of the level observed for mouse APP in non-transgenic mice. The relative expression values were determined in 3 independent Western-blot analyses using homogenates of brains from 4 homozygous ES5007 and 4 non-transgenic mice. The levels of m/hAPP protein expression ranged from 62% to 130% of control mouse APP depending on the protocol. In one experiment, APP was immunoprecipitated from equal amounts of brain homogenates from non-transgenic and homozygous ES5007 mice using PAb 369. For the other two Western-blot analyses, MAb 4G8 was used to immunoprecipitate APP. In all Western- blots, mouse APP and Swedish-FAD m/hAPP were visualized using MAb 22C11 as the detection antibody.

Processing of C-terminal domain of APP

The Swedish FAD mutation significantly altered the proteolytic processing of the of APP resulting in a change in theC-terminal fragments of

APP. The observed changes in processing was consistent with a predominat usage of the beta-secretase site over the alpha-secretase site.

Membrane preparations from brain homogenates were solubilized by detergents and APP holoprotein and C-terminal fragments were immunoprecipitated using MAb 4G8. Mouse APP and m/hAPP holoproteins were detected by Western-blot analysis using MAb 22C11. The C-terminal fragments from both mouse APP and m/hAPP were detected using PAb369 while C-terminal fragments derived exclusively from m/hAPP were detected using human specific MAb 6E10. The expression level of m/hAPP in homozygous ES5007 (Swed-homoz) brain was found to be approximately 62% of the level observed for mouse APP.

Under normal conditions, the proteolytic processing of mouse APP resulted in the generation of 5 C-terminal fragments. This contrasts with the pattern observed with Swedish-FAD m/hAPP where only the two largest C- terminal fragments were observed. The second largest C-terminal fragment (fragment 2) co-migrated with the LECIOO standard. The electrophoretic mobility of LECIOO was expected to closely resemble that of the C-terminal fragment released after the cleavage by beta-secretase. LECIOO consists of amino acid residues Leu, Gly, and Met juxtaposed with the beta-amyloid, transmembrane, and cytoplasmic domains of APP. spLEClOO (sp designates APP signal peptide, see below) was stably expressed in HEK293 cells (obtained from Sandra Reuter), a membrane homogenate prepared, and an aliquot was applied to the gel. In cells, LECIOO is generated after the signal peptide (sp) is proteolytically removed from spLEClOO during protein translation.

For other aspects of the nucleic acids, polypeptides, antibodies, etc., reference is made to standard textbooks of molecular biology, protein science, and immunology. See, e.g., Davis et al. (1986), Basic Methods in

Molecular Biology, Elsevir Sciences Publishing, Inc., New York; Hames et al. (1985), Nucleic Acid Hybridization, IL Press, Molecular Cloning, Sambrook et al.; Current Protocols in Molecular Biology, Edited by F.M. Ausubel et al., John Wiley & Sons, Inc; Current Protocols in Human Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc.;

Current Protocols in Protein Science; Edited by John E. Coligan et al., John Wiley & Sons, Inc.; Current Protocols in Immunology; Edited by John E. Coligan et al., John Wiley & Sons, Inc. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The entire disclosure of all applications, patents and publications, cited below are hereby incoφorated by reference.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Tables

Table I

Table II

n.d.: not determined

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: WIRAK, Dana O.

(ii) TITLE OF INVENTION: METHOD OF INTRODUCING MODIFICATIONS INTO A GENE

(iii) NUMBER OF SEQUENCES: 36

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Bayer Corporation

(B) STREET: 400 Morgan Lane

(C) CITY: West Haven

(D) STATE: CT

(E) COUNTRY: US

(F) ZIP: 06516-4175

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/698,360

(B) FILING DATE: 15-AUG-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Jones, Huw R.

(B) REGISTRATION NUMBER: 33,916

(C) REFERENCE/DOCKET NUMBER: WH 5009-PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (203) 812-2317

(B) TELEFAX: (203) 812-5492

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Adaptor"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : TCGACGACTT AAGTTGATAT CCACCATGGT GACGCGTT 38

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Adaptor"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

TCGAGTGAGA TCTTAAGGCC TGG 23

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Adaptor"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TCGACAAGGC GCGCCGTTTA AACAAGCGGC CGCTTGGCGC GCCTTTTGTT TAAACTTG 58

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CCTCGGCCTT TGGTGTGTGT TTTATGACAT GACCCCCTTG A 41

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CACCCTGTTG TCAATGCCTC TGGGTTTCCG CCAGTTTCG 39

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

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(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

CGATGGGTAG TGAAGCA 17

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : GTGAAGATGG ATGCAGAATT C 21

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :

GTTCTGGGCT GACAAACATC 20

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GATGGCGGAC TTCAAATCCT G 21 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CTAGACACTC 10

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ACTTTGTGTT TGACGC 16

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GATGATGAAC TTCATATCCT G 21

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CAGTTTTTGA TGGCGG 16

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GTTTGAGACC TTCAACACCC 20

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GAAGGAAGGC TGGAAAAGAG CC 22

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 770 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30

Gin lie Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys lie Asp 50 55 60

Thr Lys Glu Gly lie Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

Gin lie Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr lie Gin Asn 85 90 95

Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110 lie Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gin Glu Arg Met Asp Val Cys 130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly lie 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser lie 260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285

Glu Val Cys Ser Glu Gin Ala Glu Thr Gly Pro Cys Arg Ala Met lie 290 295 300

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gin Ser Leu Leu Lys Thr 340 345 350

Thr Gin Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380

Glu Asn Glu His Ala His Phe Gin Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400

Lys His Arg Glu Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala 405 410 415

Glu Arg Gin Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val lie 420 425 430

Gin His Phe Gin Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn 435 440 445 Glu Arg Gin Gin Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr lie Thr Ala Leu 465 470 475 480

Gin Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495

Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe 500 505 510

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gin lie Arg Ser 515 520 525

Gin Val Met Thr His Leu Arg Val lie Tyr Glu Arg Met Asn Gin Ser 530 535 540

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu lie Gin Asp 545 550 555 560

Glu Val Asp Glu Leu Leu Gin Lys Glu Gin Asn Tyr Ser Asp Asp Val 565 570 575

Leu Ala Asn Met lie Ser Glu Pro Arg lie Ser Tyr Gly Asn Asp Ala 580 585 590

Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605

Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gin Pro Trp His Ser Phe 610 615 620

Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640

Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655

Gly Leu Thr Asn lie Lys Thr Glu Glu lie Ser Glu Val Lys Met Asp 660 665 670

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu 675 680 685

Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala lie lie Gly 690 695 700

Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr Leu 705 710 715 720

Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val Val 725 730 735 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750

Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met 755 760 765

Gin Asn 770

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11992 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 6541..6639

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

NNNAAGCTTN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 60

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 720 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1500

NNNNTCTAGA NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1800

NNNNNNNNNN CCATGGNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1860

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1920

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1980

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2040

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2100

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2160

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2220

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2280

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2340

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2400 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2460

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2520

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2580

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2640

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2700

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2760

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2820

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2880

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 2940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3060

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3180

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GATCCNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8220

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NNNNNNNNNN NNNNNNNNNN GGCGCCNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9600

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NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN CCATGGNNNN NNNNNNNNNN NNNNNNNNNN 10200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10320

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NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNTCTAGANN 10500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNAGATCT NNNNNNNNNN 10560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10680

NAAGCTTNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNAG ATCTNNNNNN NNNNNNNNNN 10800 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10860

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10920

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10980

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11040

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11100

NNNNNNNNNN NNNNNNNNNN NNNNNNNNGA TATCNNNNNN NNNNNNNNNN NNNNNNNNNN 11160

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11220

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11280

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11340

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11400

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11460

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11520

NNNNNNNNNN NNNNNNNNNN NNCATATGNN NNGAATTCNN NNNNNNNNNN NNNNNNNNNN 11580

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11640

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11700

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNGT ATACNNNNNN 11760

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NGGTACCNNN NNNNNNNNNN 11820

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11880

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNCAGC 11940

TGNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNCCAT GG 11992

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12814 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: Swedish-FAD APP

(ix) FEATURE:

(A) NAME/KEY: mat_jpeptide

(B) LOCATION: 1932..2276

(D) OTHER INFORMATION: /standard_name= "Swedish-FAD APP"

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 5360..6160

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

GAGCTCCACC GCGGTGGCGG CCGCTCTGAC CATGGNNNNN NNNNNNNNNN NNNAAGCTTN 60

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 180

NNNNNNNNNN NNNNNNNNNN NCATATGNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNGAA TTCNNNNNNN 720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNG AATTCNNNNN 960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNGAATTCN 1260 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1800

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1860

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 1920

NNNNNNNNNG TTCTGGGCTG ACAAACATCA AGACGGAAGA GATCTCTGAA GTGAATCTAG 1980

ATGCAGAATT CCGACATGAC TCAGGATATG AAGTTCATCA TCAAAAATTG GTGTTCTTTG 2040

CAGAAGATGT GGGTTCAAAC AAAGGTGCAA TCATTGGACT CATGGTGGGC GGTGTTGTCA 2100

TAGCGACAGT GATCGTCATC ACCTTGGTGA TGCTGAAGAA GAAACAGTAC ACATCCATTC 2160

ATCATGGTGT GGTGGAGGTT GACGCCGCTG TCACCCCAGA GGAGCGCCAC CTGTCCAAGA 2220

TGCAGCAGAA CGGCTACGAA AATCCAACCT ACAAGTTCTT TGAGCAGATG CAGAACTAGA 2280

CCCCCGCCAC AGCAGCCTCT GAAGTTGGAC AGCAAAACCA TTGCTTCACT ACCCATCGGT 2340

GTCCATTTAT AGAATAATGT GGGAAGAAAC AAACCCGTTT TATGATTTAC TCATTATCGC 2400

CTTTTGACAG CTGTGCTGTA ACACAAGTAG ATGCCTGAAC TTGAATTAAT CCACACATCA 2460

GTAATGTATT CTATCTCTCT TTACATTTTG GTCTCTATAC TACATTATTA ATGGGTTTTG 2520

TGTACTGTAA AGAATTTAGC TGTATCAAAC TAGTGCATGA ATAGATTCTC TCCTGATTAT 2580

TTATCACATA GCCCCTTAGC CAGTTGTATA TTATTCTTGT GGTTTGTGAC CCAATTAAGT 2640

CCTACTTTAC ATATGCTTTA AGAATCGATG GGGGATGCTT CATGTGAACG TGGGAGTTCA 2700

GCTGCTTCTC TTGCCTAAGT ATTCCTTTCC TGATCACTAT GCATTTTAAA GTTAAACATT 2760

TTTAAGTATT TCAGATGCTT TAGAGAGATT TTTTTTCCAT GACTGCATTT TACTGTACAG 2820 ATTGCTGCTT CTGCTATATT TGTGATATAG GAATTAAGAG GATACACACG TTTGTTTCTT 2880 CGTGCCTGTT TTATGTGCAC ACATTAGGCA TTGAGACTTC AAGCTTTTCT TTTTTTGTCC 2940 ACGTATCTTT GGGTCTTTGA TAAAGAAAAG AATCCCTGTT CATTGTAAGC ACTTTTACGG 3000

GGCGGGTGGG GAGGGGTGCT CTGCTGGTCT TCAATTACCA AGAATTCTCC AAAACAATTT 3060

TCTGCAGGAT GATTGTACAG AATCATTGCT TATGACATGA TCGCTTTCTA CACTGTATTA 3120

CATAAATAAA TTAAATAAAA TAACCCCGGG CAAGACTTTT CTTTGAAGGA TGACTACAGA 3180

CATTAAATAA TCGAAGTAAT TTTGGGTGGG GAGAAGAGGC AGATTCAATT TTCTTTAACC 3240

AGTCTGAAGT TTCATTTATG ATACAAAAGA AGATGAAAAT GGAAGTGGCA ATATAAGGGG 3300

ATGAGGAAGG CATGCCTGGA CAAACCCTTC TTTTAAGATG TGTCTTCAAT TTGTATAAAA 3360

TGGTGTTTTC ATGTAAATAA ATACATTCTT GGAGGAGCCA CATTGTGCTG GTGTGAATGA 3420

TTCCATAGTA ACAATCTTGA CCATTTACTG ACGTACAGAC CAGTGAGAAG TCTTCGCATG 3480

TTGGGTACCC ACACCTGTTG TGTCTTAATT GCAAGTCTGA GTAGGAAGTT GGGGCCAACA 3540

TGTGTCTCCC AGTGCTGGGA AAATATTTCA TAGACCTAAT TTACAGTCTT TACTTGATCT 3600

AAAACATTTT GCTGCCATAT TTTGGCCCTC AAGTTTGTCC CAAATGAGAG ACAAAGGGAA 3660

AAGTTCCAGG GAAATAAAAA TTAAGACAGC TGATTATCTG TAAAGCATGG TTTCTCATCC 3720

TGAACGCTAC TAACATTTTG CAGGGAATAA TTCCTTGTTG AAGGGAGTTG TCCTGACCAG 3780

TGTAGGATAT TTATTTATTT TATTTATGTT TTTTGAGACG GAGTCTCGCT CTGTCACCCA 38 0

GGCTGGAGTG CAGTGGCACA ATCTCGGCTC ACTGCAAGCT CCGCCTCCCG GGTTCACGCC 3900

ATTCTCCTGC CTCAGCCTCC TGAATAGCTG GGACTCTAGG TGCCCGCCAC CACGCCCGGC 3960

TAATTTTTTG TATTTTTAGT AGAGACGGGG TTTCACCGTG TTAGCCAGGA CAGTCTTGGT 4020

CTCCTGACCT CGTGATCTGC CTGCCTCGGC CTCCCAAAGT GCTGAGATTA CAGGCGTGCA 4080

AGCCGCGCCC AGCCAGTGCT CTCCTTTTAA AAGTAGCCCA TTGGCTGGGC GCAGTGGCTC 4140

ACGCCTGTAA TCCCAGCACT TTGGGAGGCT GAGGCGGGTG GATCACGAGG TCAGGAGATC 4200

AAGAATATCC TGGCCAATAT GGTGAAACCC CATCTCTACT AAAAATACAA AAAAAAAAAA 4260

AAAAAAAAAA AGGCCGGGCA TGGTGGCGGG CGCTTGTAGT CCCAGCTACT CAGGAGGCTG 4320

AGGCAGGAGA ATGGTGTGCA CCTGGGAGGC GGAGGTTGCA GTGAGCTGAG ATCGCGCCAC 4380

TGCACTCCAG CCTGGGAGAC AGAGCGAGAC TCCGTCTCAA TAAATAAATA AATAAATAAA 4440

TAAAAGGAGG GCCTGGCACG AATGACATGC AGGGAAGGCA GTGAGCAGGT GGAGGTCCCT 4500

GTACTCGTTG TGGTGCCTTA TCTACCAGGC GGTTGAGTTG ACGTCTTTGT GGACAGAATT 4560

CGAGCTCGGT ACCCGGGGAT CCTCTAGAGT CGACCTTAAG GATATCCTTA AGGTCGACGG 4620 TATCGATAAG CTTGGGCTTG AACATCGAGC GCCAGGGCTC CGTAAAGCTA CTAGAGCACA 4680

GGCGGTGCCC CAACGTCCTG GGGCCTCTCC ACTAATAACG GCTACTTCCA ATTGATTGGA 4740

CGCGCCATCT TGCCTGCCTT ATGCATATTC AGCGGTGAAC TGAATATTCA TGAACGAGGC 4800

CCGTCCCGTC CCTCCCTCCT TCCCCCCACC CCCGGAACCC GCTCCGGAGG ACCCGAAGGG 4860

CCCCGCCTTC ATTACCGATG CGTAGGACAA ACCATTTTCC CGATGTGTGT GGGGGGATAC 4920

TAATGAGAGA CTTTAGCTGA AAAATGAGCC TGAACTCCGA AGCTGAGTAA AAATGGCCTA 4980

ACTTTATCCT CCGTTCTGTA AGTCCTCGGT TTGAGTGCAC GGGAAACCCG AAAGGAGGAC 5040

GACAGGACCA GGACATTCTC CTCCTCCTGT CGCGTCAGAA AGAACACCCA ACCAGGGAGC 5100

CGGAGCCCTA GCGTCAACAA CTCCGCCGCG CGCGCTCCGT GTAGGCCGGT GCGGGCGGCC 5160

CCGTAGCGCA AGGGAGGGCG GGAAAGGAAG GGGCGGGACA CAAGGGCGAA TCTATAAAGG 5220

GCGTCACTCA GCCAGTTCTC TCCTCAGAAG CGCCGAGAGC GCGACCGGGA CGGTTGGAGA 5280

AGAAGGTGGC TCCCGGAAGG GGGAGAGACA AACTGCCGTA ACCTCTGCCG TTCAGGATCA 5340

TCGAATTCCT GCAGCCAATA TGGGATCGGC CATTGAACAA GATGGATTGC ACGCAGGTTC 5400

TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA CAATCGGCTG 5460

CTCTGATGCC GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC 5520

CGACCTGTCC GGTGCCCTGA ATGAACTGCA GGACGAGGCA GCGCGGCTAT CGTGGCTGGC 5580

CACGACGGGC GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG 5640

GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA 5700

GAAAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC CGGCTACCTG 5760

CCCATTCGAC CACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG 5820

TCTTGTCGAT CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT 5880

CGCCAGGCTC AAGGCGCGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC ATGGCGATGC 5940

CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG 6000

GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA 6060

GCTTGGCGGC GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTCCCGATTC 6120

GCAGCGCATC GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGGGGATC AATTCTCTAG 6180

AGCTCGCTGA TCAGCCTCGA CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC 6240

CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA 6300 GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA 6360

GGACAGCAAG GGGGAGGATT GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC 6420

TATGGCTTCT GAGGCGGAAA GAACCAGCTG GGGCTCGAGA GATCTTCACA ANGATAGGAA 6480

GGAGAGGAAG TGGGGCTCTG TTGATAGTTC TTGCTGAGCA GAAGCCNNNN NNNNNNNNNN 6540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 6960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7800

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNG 7860

GATCCNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7920

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 7980 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8040

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8100

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8160

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8220

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8280

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8340

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8400

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8460

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8520

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8580

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8640

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8700

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8760

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8820

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8880

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 8940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9060

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9240

NNNNNNNNNN NNNNNNNNNN GGCGCCNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9660 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NCCATGGTCT AGAACTAGTG GATCCCCCGG 9900

GCTGCAGGAA TTCGATATCA AGCTTATCGA TACCGTCGAC CTCGAGGGGG GGCCCGGTAC 9960

CCAATTCGCC CTATAGTGAG TCGTATTACG CGCGCTCACT GGCCGTCGTT TTACAACGTC 10020

GTGACTGGGA AAACCCTGGC GTTACCCAAC TTAATCGCCT TGCAGCACAT CCCCCTTTCG 10080

CCAGCTGGCG TAATAGCGAA GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC 10140

TGAATGGCGA ATGGGACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA 10200

CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC 10260

CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT 10320

TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT TAGGGTGATG 10380

GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA 10440

CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT 10500

ATTCTTTTGA TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA 10560

TTTAACAAAA ATTTAACGCG AATTTTAACA AAATATTAAC GCTTACAATT TAGGTGGCAC 106 0

TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT 10680

GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG 10740

TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC 10800

TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC 10860

ACGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC 10920

CGAAGAACGT TTTCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC 10980

CCGTATTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT 11040

GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT 11100

ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT 11160

CGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT 11220

TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT 11280

GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC 11340 TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG 11400

CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC 11460

TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA 11520

CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC 11580

CTCACTGATT AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA 11640

TTTAAAACTT CATTTTTAAT TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT 11700

GACCAAAATC CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT 11760

CAAAGGATCT TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA 11820

ACCACCGCTA CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA 11880

GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT 11940

AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT 12000

ACCAGTGGCT GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA 12060

GTTACCGGAT AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT 12120

GGAGCGAACG ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC 12180

GCTTCCCGAA GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA 12240

GCGCACGAGG GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG 12300

CCACCTCTGA CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA 12360

AAACGCCAGC AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT 12420

GTTCTTTCCT GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC 12480

TGATACCGCT CGCCGCAGCC GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA 12540

AGAGCGCCCA ATACGCAAAC CGCCTCTCCC CGCGCGTTGG CCGATTCATT AATGCAGCTG 12600

GCACGACAGG TTTCCCGACT GGAAAGCGGG CAGTGAGCGC AACGCAATTA ATGTGAGTTA 12660

GCTCACTCAT TAGGCACCCC AGGCTTTACA CTTTATGCTT CCGGCTCGTA TGTTGTGTGG 12720

AATTGTGAGC GGATAACAAT TTCACACAGG AAACAGCTAT GACCATGATT ACGCCAAGCG 12780

CGCAATTAAC CCTCACTAAA GGGAACAAAA GCTG 12814

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15692 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: London-FAD APP

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 4807..5151

(ix) FEATURE:

(A) NAME/KEY: mutation

(B) LOCATION: replace (4990 , "")

(D) OTHER INFORMATION: /standard_name= "London-FAD"

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 8223..9023

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

GAGCTCCACC GCGGTGGCGG CCGCTCTAGA ACTAGTGGAT CCCCCGGGCT GCAGGAATTC 60

TACCGGGGTA GGGGAGGCGC TTTTCCCAAG GCAGTCTGGA GCATGCGCTT TAGCAGCCCC 120

GCTGGCACTT GGCGCTACAC AAGTGGCCTC TGGCCTCGCA CACATTCCAC ATCCACCGGT 180

AGCGCCAACC GGCTCCCTTC TTTGGTGGCC CCTTCGCGCC ACCTTCTACT CCTCCCCTAG 240

TCAGGAAGTT CCCCCCCGCC CCGCAGCTCG CGTCGTGCAG GACGTGACAA ATGGAAGTAG 300

CACGTCTCAC TAGTCTCGTG CAGATGGACA GCACCGCTGA GCAATGGAAG CGGGTAGGCC 360

TTTGGGGCAG CGGCCAATAG CAGCTTTGCT CCTTCGCTTT CTGGGCTCAG AGGCTGGGAA 420

GGGGTGGGTC CGGGGGCGGG CTCAGGGGCG GGCTCAGGGG CGGGGCGGGC GCGAAGGTCC 480

TCCGGAGCCC GGCATTCTGC ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT 540

CCTCATCTCC GGGCCTTTCG ACCTGCAGCG ACCCGCTTAA CAGCGTCAAC AGCGTGCCGC 600

AGATCTTGGT GGCGTGAAAC TCCCGCACCT CTTTGGCAAG CGCCTTGTAG AAGCGCGTAT 660

GGCTTCGTAC CCCTGCCATC AACACGCGTC TGCGTTCGAC CAGGCTGCGC GTTCTCGCGG 720

CCATAGCAAC CGACGTACGG CGTTGCGCCC TCGCCGGCAG CAAGAAGCCA CGGAAGTCCG 780

CCTGGAGCAG AAAATGCCCA CGCTACTGCG GGTTTATATA GACGGTCCTC ACGGGATGGG 840 GAAAACCACC ACCACGCAAC TGCTGGTGGC CCTGGGTTCG CGCGACGATA TCGTCTACGT 900

ACCCGAGCCG ATGACTTACT GGCAGGTGCT GGGGGCTTCC GAGACAATCG CGAACATCTA 960

CACCACACAA CACCGCCTCG ACCAGGGTGA GATATCGGCC GGGGACGCGG CGGTGGTAAT 1020

GACAAGCGCC CAGATAACAA TGGGCATGCC TTATGCCGTG ACCGACGCCG TTCTGGCTCC 1080

TCATGTCGGG GGGGAGGCTG GGAGTTCACA TGCCCCGCCC CCGGCCCTCA CCCTCATCTT 1140

CGACCGCCAT CCCATCGCCG CCCTCCTGTG CTACCCGGCC GCGCGATACC TTATGGGCAG 1200

CATGACCCCC CAGGCCGTGC TGGCGTTCGT GGCCCTCATC CCGCCGACCT TGCCCGGCAC 1260

AAACATCGTG TTGGGGGCCC TTCCGGAGGA CAGACACATC GACCGCCTGG CCAAACGCCA 1320

GCGCCCCGGC GAGCGGCTTG ACCTGGCTAT GCTGGCCGCG ATTCGCCGCG TTTACGGGCT 1380

GCTTGCCAAT ACGGTGCGGT ATCTGCAGGG CGGCGGGTCG TGGTGGGAGG ATTGGGGACA 14 0

GCTTTCGGGG ACGGCCGTGC CGCCCCAGGG TGCCGAGCCC CAGAGCAACG CGGGCCCACG 1500

ACCCCATATC GGGGACACGT TATTTACCCT GTTTCGGGCC CCCGAGTTGC TGGCCCCCAA 1560

CGGCGACCTG TATAACGTGT TTGCCTGGGC CTTGGACGTC TTGGCCAAAC GCCTCCGTCC 1620

CATGCACGTC TTTATCCTGG ATTACGACCA ATCGCCCGCC GGCTGCCGGG ACGCCCTGCT 1680

GCAACTTACC TCCGGGATGG TCCAGACCCA CGTCACCACC CCAGGCTCCA TACCGACGAT 1740

CTGCGACCTG GCGCGCACGT TTGCCCGGGA GATGGGGGAG GCTAACTGAA ACACGGAAGG 1800

AGACAATACC GGAAGGAACC CGCGCTATGA CGGCAATAAA AAGACAGAAT AAAACGCACG 1860

GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT 1920

ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAA 1980

CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AAGCCCGCCA 2040

TAGCCACGGG CCCCGTGGGT TAGGGACGGG GTCCCCCATG GGGAATGGTT TATGGTTCGT 2100

GGGGGTTATT CTTTTGGGCG TTGCGTGGGG TCAGGTCCAC GACTGGACTG AGCAGACAGA 2160

CCCATGGTTT TTGGATGGCC TGGGCATGGA CCGCATGTAC TGGCGCGACA CGAACACCGG 2220

GCGTCTGTGG CTGCCAAACA CCCCCGACCC CCAAAAACCA CCGCGCGGAT TTCTGGCGCC 2280

GCCGGACGAA CTAAACCTGA CTACGGCATC TCTGCCCCTT CTTCGCTGGT ACGAGGAGCG 2340

CTTTTGTTTT GTATTGGTCA CCACGGCCGA GTTTCCGCGG GACCCCGGCC AGGACCTGCA 2400

GAAATTGATG ATCTATTAAA CAATAAAGAT GTCCACTAAA ATGGAAGTTT TTTCCTGTCA 2460

TACTTTGTTA AGAAGGGTGA GAACAGAGTA CCTACATTTT GAATGGAAGG ATTGGAGCTA 2520 CGGGGGTGGG GGTGGGGTGG GATTAGATAA ATGCCTGCTC TTTACTGAAG GCTCTTTACT 2580

ATTGCTTTAT GATAATGTTT CATAGTTGGA TATCATAATT TAAACAAGCA AAACCAAATT 2640

AAGGGCCAGC TCATTCCTCC ACTCATGATC TATAGATCTA TAGATCTCTC GTGGGATCAT 2700

TGTTTTTCTC TTGATTCCCA CTTTGTGTTC TAAGTACTGT GGTTTCCAAA TGTGTCAGTT 2760

TCATAGCCTG AAGAACGAGA TCAGCAGCCT CTGTTCCACA TACACTTCAT TCTCAGTATT 2820

GTTTTGCCAA GTTCTAATTC CATCAGATCA AGCTTATCGA TACCGTCGAC AAGGCGCGCC 2880

ATGTTTAAAC TTGCGGCCGC TCTGACCATG GNNNNNNNNN NNNNNNNNNA AGCTTNNNNN 2940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3060

NNNNNNNNNN NNNNNNNCAT ATGNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNGAATTCN NNNNNNNNNN 3600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNGAATT CNNNNNNNNN 3840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNG AATTCNNNNN 4140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4200 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4800

NNNNGTTCTG GGCTGACAAA CATCAAGACG GAAGAGATCT CTGAAGTGAA GATGGATGCA 4860

GAATTCCGAC ATGACTCAGG ATATGAAGTT CATCATCAAA AATTGGTGTT CTTTGCAGAA 4920

GATGTGGGTT CAAACAAAGG TGCAATCATT GGACTCATGG TGGGCGGTGT TGTCATAGCG 4980

ACAGTGATAA TCATCACCTT GGTGATGCTG AAGAAGAAAC AGTACACATC CATTCATCAT 5040

GGTGTGGTGG AGGTTGACGC CGCTGTCACC CCAGAGGAGC GCCACCTGTC CAAGATGCAG 5100

CAGAACGGCT ACGAAAATCC AACCTACAAG TTCTTTGAGC AGATGCAGAA CTAGACCCCC 5160

GCCACAGCAG CCTCTGAAGT TGGACAGCAA AACCATTGCT TCACTACCCA TCGGTGTCCA 5220

TTTATAGAAT AATGTGGGAA GAAACAAACC CGTTTTATGA TTTACTCATT ATCGCCTTTT 5280

GACAGCTGTG CTGTAACACA AGTAGATGCC TGAACTTGAA TTAATCCACA CATCAGTAAT 5340

GTATTCTATC TCTCTTTACA TTTTGGTCTC TATACTACAT TATTAATGGG TTTTGTGTAC 5400

TGTAAAGAAT TTAGCTGTAT CAAACTAGTG CATGAATAGA TTCTCTCCTG ATTATTTATC 5460

ACATAGCCCC TTAGCCAGTT GTATATTATT CTTGTGGTTT GTGACCCAAT TAAGTCCTAC 5520

TTTACATATG CTTTAAGAAT CGATGGGGGA TGCTTCATGT GAACGTGGGA GTTCAGCTGC 5580

TTCTCTTGCC TAAGTATTCC TTTCCTGATC ACTATGCATT TTAAAGTTAA ACATTTTTAA 5640

GTATTTCAGA TGCTTTAGAG AGATTTTTTT TCCATGACTG CATTTTACTG TACAGATTGC 5700

TGCTTCTGCT ATATTTGTGA TATAGGAATT AAGAGGATAC ACACGTTTGT TTCTTCGTGC 5760

CTGTTTTATG TGCACACATT AGGCATTGAG ACTTCAAGCT TTTCTTTTTT TGTCCACGTA 5820

TCTTTGGGTC TTTGATAAAG AAAAGAATCC CTGTTCATTG TAAGCACTTT TACGGGGCGG 5880 GTGGGGAGGG GTGCTCTGCT GGTCTTCAAT TACCAAGAAT TCTCCAAAAC AATTTTCTGC 5940

AGGATGATTG TACAGAATCA TTGCTTATGA CATGATCGCT TTCTACACTG TATTACATAA 6000

ATAAATTAAA TAAAATAACC CCGGGCAAGA CTTTTCTTTG AAGGATGACT ACAGACATTA 6060

AATAATCGAA GTAATTTTGG GTGGGGAGAA GAGGCAGATT CAATTTTCTT TAACCAGTCT 6120

GAAGTTTCAT TTATGATACA AAAGAAGATG AAAATGGAAG TGGCAATATA AGGGGATGAG 6180

GAAGGCATGC CTGGACAAAC CCTTCTTTTA AGATGTGTCT TCAATTTGTA TAAAATGGTG 6240

TTTTCATGTA AATAAATACA TTCTTGGAGG AGCCACATTG TGCTGGTGTG AATGATTCCA 6300

TAGTAACAAT CTTGACCATT TACTGACGTA CAGACCAGTG AGAAGTCTTC GCATGTTGGG 6360

TACCCACACC TGTTGTGTCT TAATTGCAAG TCTGAGTAGG AAGTTGGGGC CAACATGTGT 6420

CTCCCAGTGC TGGGAAAATA TTTCATAGAC CTAATTTACA GTCTTTACTT GATCTAAAAC 6480

ATTTTGCTGC CATATTTTGG CCCTCAAGTT TGTCCCAAAT GAGAGACAAA GGGAAAAGTT 6540

CCAGGGAAAT AAAAATTAAG ACAGCTGATT ATCTGTAAAG CATGGTTTCT CATCCTGAAC 6600

GCTACTAACA TTTTGCAGGG AATAATTCCT TGTTGAAGGG AGTTGTCCTG ACCAGTGTAG 6660

GATATTTATT TATTTTATTT ATGTTTTTTG AGACGGAGTC TCGCTCTGTC ACCCAGGCTG 6720

GAGTGCAGTG GCACAATCTC GGCTCACTGC AAGCTCCGCC TCCCGGGTTC ACGCCATTCT 6780

CCTGCCTCAG CCTCCTGAAT AGCTGGGACT CTAGGTGCCC GCCACCACGC CCGGCTAATT 6840

TTTTGTATTT TTAGTAGAGA CGGGGTTTCA CCGTGTTAGC CAGGACAGTC TTGGTCTCCT 6900

GACCTCGTGA TCTGCCTGCC TCGGCCTCCC AAAGTGCTGA GATTACAGGC GTGCAAGCCG 6960

CGCCCAGCCA GTGCTCTCCT TTTAAAAGTA GCCCATTGGC TGGGCGCAGT GGCTCACGCC 7020

TGTAATCCCA GCACTTTGGG AGGCTGAGGC GGGTGGATCA CGAGGTCAGG AGATCAAGAA 7080

TATCCTGGCC AATATGGTGA AACCCCATCT CTACTAAAAA TACAAAAAAA AAAAAAAAAA 7140

AAAAAAGGCC GGGCATGGTG GCGGGCGCTT GTAGTCCCAG CTACTCAGGA GGCTGAGGCA 7200

GGAGAATGGT GTGCACCTGG GAGGCGGAGG TTGCAGTGAG CTGAGATCGC GCCACTGCAC 7260

TCCAGCCTGG GAGACAGAGC GAGACTCCGT CTCAATAAAT AAATAAATAA ATAAATAAAA 7320

GGAGGGCCTG GCACGAATGA CATGCAGGGA AGGCAGTGAG CAGGTGGAGG TCCCTGTACT 7380

CGTTGTGGTG CCTTATCTAC CAGGCGGTTG AGTTGACGTC TTTGTGGACA GAATTCGAGC 7440

TCGGTACCCG GGGATCCTCT AGAGTCGACC TTAAGGTCGA CGGTATCGAT AAGCTTGGGC 7500

TTGAACATCG AGCGCCAGGG CTCCGTAAAG CTACTAGAGC ACAGGCGGTG CCCCAACGTC 7560 CTGGGGCCTC TCCACTAATA ACGGCTACTT CCAATTGATT GGACGCGCCA TCTTGCCTGC 7620

CTTATGCATA TTCAGCGGTG AACTGAATAT TCATGAACGA GGCCCGTCCC GTCCCTCCCT 7680

CCTTCCCCCC ACCCCCGGAA CCCGCTCCGG AGGACCCGAA GGGCCCCGCC TTCATTACCG 7740

ATGCGTAGGA CAAACCATTT TCCCGATGTG TGTGGGGGGA TACTAATGAG AGACTTTAGC 7800

TGAAAAATGA GCCTGAACTC CGAAGCTGAG TAAAAATGGC CTAACTTTAT CCTCCGTTCT 7860

GTAAGTCCTC GGTTTGAGTG CACGGGAAAC CCGAAAGGAG GACGACAGGA CCAGGACATT 7920

CTCCTCCTCC TGTCGCGTCA GAAAGAACAC CCAACCAGGG AGCCGGAGCC CTAGCGTCAA 7980

CAACTCCGCC GCGCGCGCTC CGTGTAGGCC GGTGCGGGCG GCCCCGTAGC GCAAGGGAGG 8040

GCGGGAAAGG AAGGGGCGGG ACACAAGGGC GAATCTATAA AGGGCGTCAC TCAGCCAGTT 8100

CTCTCCTCAG AAGCGCCGAG AGCGCGACCG GGACGGTTGG AGAAGAAGGT GGCTCCCGGA 8160

AGGGGGAGAG ACAAACTGCC GTAACCTCTG CCGTTCAGGA TCATCGAATT CCTGCAGCCA 8220

ATATGGGATC GGCCATTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG 8280

AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT 8340

TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC 8400

TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT 8460

GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG 8520

TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG 8580

CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG 8640

CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG 8700

ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC 8760

GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA 8820

TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC 8880

GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG 8940

CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT 9000

ATCGCCTTCT TGACGAGTTC TTCTGAGGGG ATCAATTCTC TAGAGCTCGC TGATCAGCCT 9060

CGACTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA 9120

CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT 9180

GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG 9240 ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG 9300

AAAGAACCAG CTGGGGCTCG AGAGATCTTC ACAANGATAG GAAGGAGAGG AAGTGGGGCT 9360

CTGTTGATAG TTCTTGCTGA GCAGAAGCCN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9480 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9540 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9600 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9660 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9720 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9780 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9840 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9900 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNGG 10740

ATCCNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10800

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10860

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10920 - Ill -

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10980

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11040

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11100

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11160

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11220

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11280

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11340

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11400

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11460

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11520

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11580

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11640

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11700

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11760

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11820

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11880

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12060

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12120

NNNNNNNNNN NNNNNNNNNG GCGCCNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12600 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNC CATGGTCTAG AACTAGTGGA TCCCCCGGGC 12780

TGCAGGAATT CGATATCAAG CTTATCGATA CCGTCGACCT CGAGGGGGGG CCCGGTACCC 12840

AATTCGCCCT ATAGTGAGTC GTATTACGCG CGCTCACTGG CCGTCGTTTT ACAACGTCGT 12900

GACTGGGAAA ACCCTGGCGT TACCCAACTT AATCGCCTTG CAGCACATCC CCCTTTCGCC 12960

AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT CCCAACAGTT GCGCAGCCTG 13020

AATGGCGAAT GGGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG 13080

CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT 13140

TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG GCTCCCTTTA 13200

GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT 13260

TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG 13320

TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT 13380

TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGCCTATT GGTTAAAAAA TGAGCTGATT 13440

TAACAAAAAT TTAACGCGAA TTTTAACAAA ATATTAACGC TTACAATTTA GGTGGCACTT 13500

TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT 13560

ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA 13620

TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG 13680

TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC TGAAGATCAG TTGGGTGCAC 13740

GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG 13800

AAGAACGTTT TCCAATGATG AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC 13860

GTATTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG 13920

TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT 13980

GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA CTTACTTCTG ACAACGATCG 14040

GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG GGATCATGTA ACTCGCCTTG 14100

ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC 14160

CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT 14220

CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT 14280 CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG AGCCGGTGAG CGTGGGTCTC 14340

GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC CCGTATCGTA GTTATCTACA 14400

CGACGGGGAG TCAGGCAACT ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT 14460

CACTGATTAA GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT 14520

TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA 14580

CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA 14640

AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC 14700

CACCGCTACC AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG 14760

TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTCCT TCTAGTGTAG CCGTAGTTAG 14820

GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC 14880

CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA AGACGATAGT 14940

TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG 15000

AGCGAACGAC CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC 15060

TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC 15120

GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC 15180

ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC CTATGGAAAA 15240

ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT 15300

TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCTTT GAGTGAGCTG 15360

ATACCGCTCG CCGCAGCCGA ACGACCGAGC GCAGCGAGTC AGTGAGCGAG GAAGCGGAAG 15420

AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC 15480

ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTGAGTTAGC 15540

TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA 15600

TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCGCG 15660

CAATTAACCC TCACTAAAGG GAACAAAAGC TG 15692

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15692 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: Swedish/London-FAD APP

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 4807..5151

(ix) FEATURE:

(A) NAME/KEY: mutation

(B) LOCATION: replace (4849 , "")

(D) OTHER INFORMATION: /standard_name= "Swedish-FAD"

(ix) FEATURE:

(A) NAME/KEY: mutation

(B) LOCATION: replace (4989, "")

(D) OTHER INFORMATION: /standard_name= "London-FAD"

(ix) FEATURE:

(A) NAME/KEY: matjpeptide

(B) LOCATION: 8223..9023

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GAGCTCCACC GCGGTGGCGG CCGCTCTAGA ACTAGTGGAT CCCCCGGGCT GCAGGAATTC 60

TACCGGGGTA GGGGAGGCGC TTTTCCCAAG GCAGTCTGGA GCATGCGCTT TAGCAGCCCC 120

GCTGGCACTT GGCGCTACAC AAGTGGCCTC TGGCCTCGCA CACATTCCAC ATCCACCGGT 180

AGCGCCAACC GGCTCCCTTC TTTGGTGGCC CCTTCGCGCC ACCTTCTACT CCTCCCCTAG 240

TCAGGAAGTT CCCCCCCGCC CCGCAGCTCG CGTCGTGCAG GACGTGACAA ATGGAAGTAG 300

CACGTCTCAC TAGTCTCGTG CAGATGGACA GCACCGCTGA GCAATGGAAG CGGGTAGGCC 360

TTTGGGGCAG CGGCCAATAG CAGCTTTGCT CCTTCGCTTT CTGGGCTCAG AGGCTGGGAA 420

GGGGTGGGTC CGGGGGCGGG CTCAGGGGCG GGCTCAGGGG CGGGGCGGGC GCGAAGGTCC 480

TCCGGAGCCC GGCATTCTGC ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT 540

CCTCATCTCC GGGCCTTTCG ACCTGCAGCG ACCCGCTTAA CAGCGTCAAC AGCGTGCCGC 600

AGATCTTGGT GGCGTGAAAC TCCCGCACCT CTTTGGCAAG CGCCTTGTAG AAGCGCGTAT 660

GGCTTCGTAC CCCTGCCATC AACACGCGTC TGCGTTCGAC CAGGCTGCGC GTTCTCGCGG 720

CCATAGCAAC CGACGTACGG CGTTGCGCCC TCGCCGGCAG CAAGAAGCCA CGGAAGTCCG 780 CCTGGAGCAG AAAATGCCCA CGCTACTGCG GGTTTATATA GACGGTCCTC ACGGGATGGG 840

GAAAACCACC ACCACGCAAC TGCTGGTGGC CCTGGGTTCG CGCGACGATA TCGTCTACGT 900

ACCCGAGCCG ATGACTTACT GGCAGGTGCT GGGGGCTTCC GAGACAATCG CGAACATCTA 960

CACCACACAA CACCGCCTCG ACCAGGGTGA GATATCGGCC GGGGACGCGG CGGTGGTAAT 1020

GACAAGCGCC CAGATAACAA TGGGCATGCC TTATGCCGTG ACCGACGCCG TTCTGGCTCC 1080

TCATGTCGGG GGGGAGGCTG GGAGTTCACA TGCCCCGCCC CCGGCCCTCA CCCTCATCTT 1140

CGACCGCCAT CCCATCGCCG CCCTCCTGTG CTACCCGGCC GCGCGATACC TTATGGGCAG 1200

CATGACCCCC CAGGCCGTGC TGGCGTTCGT GGCCCTCATC CCGCCGACCT TGCCCGGCAC 1260

AAACATCGTG TTGGGGGCCC TTCCGGAGGA CAGACACATC GACCGCCTGG CCAAACGCCA 1320

GCGCCCCGGC GAGCGGCTTG ACCTGGCTAT GCTGGCCGCG ATTCGCCGCG TTTACGGGCT 1380

GCTTGCCAAT ACGGTGCGGT ATCTGCAGGG CGGCGGGTCG TGGTGGGAGG ATTGGGGACA 1440

GCTTTCGGGG ACGGCCGTGC CGCCCCAGGG TGCCGAGCCC CAGAGCAACG CGGGCCCACG 1500

ACCCCATATC GGGGACACGT TATTTACCCT GTTTCGGGCC CCCGAGTTGC TGGCCCCCAA 1560

CGGCGACCTG TATAACGTGT TTGCCTGGGC CTTGGACGTC TTGGCCAAAC GCCTCCGTCC 1620

CATGCACGTC TTTATCCTGG ATTACGACCA ATCGCCCGCC GGCTGCCGGG ACGCCCTGCT 1680

GCAACTTACC TCCGGGATGG TCCAGACCCA CGTCACCACC CCAGGCTCCA TACCGACGAT 1740

CTGCGACCTG GCGCGCACGT TTGCCCGGGA GATGGGGGAG GCTAACTGAA ACACGGAAGG 1800

AGACAATACC GGAAGGAACC CGCGCTATGA CGGCAATAAA AAGACAGAAT AAAACGCACG 1860

GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT 1920

ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAA 1980

CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AAGCCCGCCA 2040

TAGCCACGGG CCCCGTGGGT TAGGGACGGG GTCCCCCATG GGGAATGGTT TATGGTTCGT 2100

GGGGGTTATT CTTTTGGGCG TTGCGTGGGG TCAGGTCCAC GACTGGACTG AGCAGACAGA 2160

CCCATGGTTT TTGGATGGCC TGGGCATGGA CCGCATGTAC TGGCGCGACA CGAACACCGG 2220

GCGTCTGTGG CTGCCAAACA CCCCCGACCC CCAAAAACCA CCGCGCGGAT TTCTGGCGCC 2280

GCCGGACGAA CTAAACCTGA CTACGGCATC TCTGCCCCTT CTTCGCTGGT ACGAGGAGCG 2340

CTTTTGTTTT GTATTGGTCA CCACGGCCGA GTTTCCGCGG GACCCCGGCC AGGACCTGCA 2400

GAAATTGATG ATCTATTAAA CAATAAAGAT GTCCACTAAA ATGGAAGTTT TTTCCTGTCA 2460 TACTTTGTTA AGAAGGGTGA GAACAGAGTA CCTACATTTT GAATGGAAGG ATTGGAGCTA 2520

CGGGGGTGGG GGTGGGGTGG GATTAGATAA ATGCCTGCTC TTTACTGAAG GCTCTTTACT 2580

ATTGCTTTAT GATAATGTTT CATAGTTGGA TATCATAATT TAAACAAGCA AAACCAAATT 2640

AAGGGCCAGC TCATTCCTCC ACTCATGATC TATAGATCTA TAGATCTCTC GTGGGATCAT 2700

TGTTTTTCTC TTGATTCCCA CTTTGTGTTC TAAGTACTGT GGTTTCCAAA TGTGTCAGTT 2760

TCATAGCCTG AAGAACGAGA TCAGCAGCCT CTGTTCCACA TACACTTCAT TCTCAGTATT 2820

GTTTTGCCAA GTTCTAATTC CATCAGATCA AGCTTATCGA TACCGTCGAC AAGGCGCGCC 2880

ATGTTTAAAC TTGCGGCCGC TCTGACCATG GNNNNNNNNN NNNNNNNNNA AGCTTNNNNN 2940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3060

NNNNNNNNNN NNNNNNNCAT ATGNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNGAATTCN NNNNNNNNNN 3600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNGAATT CNNNNNNNNN 3840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNG AATTCNNNNN 4140 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4800

NNNNGTTCTG GGCTGACAAA CATCAAGACG GAAGAGATCT CTGAAGTGAA TCTAGATGCA 4860

GAATTCCGAC ATGACTCAGG ATATGAAGTT CATCATCAAA AATTGGTGTT CTTTGCAGAA 4920

GATGTGGGTT CAAACAAAGG TGCAATCATT GGACTCATGG TGGGCGGTGT TGTCATAGCG 4980

ACAGTGATAA TCATCACCTT GGTGATGCTG AAGAAGAAAC AGTACACATC CATTCATCAT 5040

GGTGTGGTGG AGGTTGACGC CGCTGTCACC CCAGAGGAGC GCCACCTGTC CAAGATGCAG 5100

CAGAACGGCT ACGAAAATCC AACCTACAAG TTCTTTGAGC AGATGCAGAA CTAGACCCCC 5160

GCCACAGCAG CCTCTGAAGT TGGACAGCAA AACCATTGCT TCACTACCCA TCGGTGTCCA 5220

TTTATAGAAT AATGTGGGAA GAAACAAACC CGTTTTATGA TTTACTCATT ATCGCCTTTT 5280

GACAGCTGTG CTGTAACACA AGTAGATGCC TGAACTTGAA TTAATCCACA CATCAGTAAT 5340

GTATTCTATC TCTCTTTACA TTTTGGTCTC TATACTACAT TATTAATGGG TTTTGTGTAC 5400

TGTAAAGAAT TTAGCTGTAT CAAACTAGTG CATGAATAGA TTCTCTCCTG ATTATTTATC 5460

ACATAGCCCC TTAGCCAGTT GTATATTATT CTTGTGGTTT GTGACCCAAT TAAGTCCTAC 5520

TTTACATATG CTTTAAGAAT CGATGGGGGA TGCTTCATGT GAACGTGGGA GTTCAGCTGC 5580

TTCTCTTGCC TAAGTATTCC TTTCCTGATC ACTATGCATT TTAAAGTTAA ACATTTTTAA 5640

GTATTTCAGA TGCTTTAGAG AGATTTTTTT TCCATGACTG CATTTTACTG TACAGATTGC 5700

TGCTTCTGCT ATATTTGTGA TATAGGAATT AAGAGGATAC ACACGTTTGT TTCTTCGTGC 5760

CTGTTTTATG TGCACACATT AGGCATTGAG ACTTCAAGCT TTTCTTTTTT TGTCCACGTA 5820 TCTTTGGGTC TTTGATAAAG AAAAGAATCC CTGTTCATTG TAAGCACTTT TACGGGGCGG 5880

GTGGGGAGGG GTGCTCTGCT GGTCTTCAAT TACCAAGAAT TCTCCAAAAC AATTTTCTGC 5940

AGGATGATTG TACAGAATCA TTGCTTATGA CATGATCGCT TTCTACACTG TATTACATAA 6000

ATAAATTAAA TAAAATAACC CCGGGCAAGA CTTTTCTTTG AAGGATGACT ACAGACATTA 6060

AATAATCGAA GTAATTTTGG GTGGGGAGAA GAGGCAGATT CAATTTTCTT TAACCAGTCT 6120

GAAGTTTCAT TTATGATACA AAAGAAGATG AAAATGGAAG TGGCAATATA AGGGGATGAG 6180

GAAGGCATGC CTGGACAAAC CCTTCTTTTA AGATGTGTCT TCAATTTGTA TAAAATGGTG 6240

TTTTCATGTA AATAAATACA TTCTTGGAGG AGCCACATTG TGCTGGTGTG AATGATTCCA 6300

TAGTAACAAT CTTGACCATT TACTGACGTA CAGACCAGTG AGAAGTCTTC GCATGTTGGG 6360

TACCCACACC TGTTGTGTCT TAATTGCAAG TCTGAGTAGG AAGTTGGGGC CAACATGTGT 6420

CTCCCAGTGC TGGGAAAATA TTTCATAGAC CTAATTTACA GTCTTTACTT GATCTAAAAC 6480

ATTTTGCTGC CATATTTTGG CCCTCAAGTT TGTCCCAAAT GAGAGACAAA GGGAAAAGTT 6540

CCAGGGAAAT AAAAATTAAG ACAGCTGATT ATCTGTAAAG CATGGTTTCT CATCCTGAAC 6600

GCTACTAACA TTTTGCAGGG AATAATTCCT TGTTGAAGGG AGTTGTCCTG ACCAGTGTAG 6660

GATATTTATT TATTTTATTT ATGTTTTTTG AGACGGAGTC TCGCTCTGTC ACCCAGGCTG 6720

GAGTGCAGTG GCACAATCTC GGCTCACTGC AAGCTCCGCC TCCCGGGTTC ACGCCATTCT 6780

CCTGCCTCAG CCTCCTGAAT AGCTGGGACT CTAGGTGCCC GCCACCACGC CCGGCTAATT 6840

TTTTGTATTT TTAGTAGAGA CGGGGTTTCA CCGTGTTAGC CAGGACAGTC TTGGTCTCCT 6900

GACCTCGTGA TCTGCCTGCC TCGGCCTCCC AAAGTGCTGA GATTACAGGC GTGCAAGCCG 6960

CGCCCAGCCA GTGCTCTCCT TTTAAAAGTA GCCCATTGGC TGGGCGCAGT GGCTCACGCC 7020

TGTAATCCCA GCACTTTGGG AGGCTGAGGC GGGTGGATCA CGAGGTCAGG AGATCAAGAA 7080

TATCCTGGCC AATATGGTGA AACCCCATCT CTACTAAAAA TACAAAAAAA AAAAAAAAAA 7140

AAAAAAGGCC GGGCATGGTG GCGGGCGCTT GTAGTCCCAG CTACTCAGGA GGCTGAGGCA 7200

GGAGAATGGT GTGCACCTGG GAGGCGGAGG TTGCAGTGAG CTGAGATCGC GCCACTGCAC 7260

TCCAGCCTGG GAGACAGAGC GAGACTCCGT CTCAATAAAT AAATAAATAA ATAAATAAAA 7320

GGAGGGCCTG GCACGAATGA CATGCAGGGA AGGCAGTGAG CAGGTGGAGG TCCCTGTACT 7380

CGTTGTGGTG CCTTATCTAC CAGGCGGTTG AGTTGACGTC TTTGTGGACA GAATTCGAGC 7440

TCGGTACCCG GGGATCCTCT AGAGTCGACC TTAAGGTCGA CGGTATCGAT AAGCTTGGGC 7500 TTGAACATCG AGCGCCAGGG CTCCGTAAAG CTACTAGAGC ACAGGCGGTG CCCCAACGTC 7560

CTGGGGCCTC TCCACTAATA ACGGCTACTT CCAATTGATT GGACGCGCCA TCTTGCCTGC 7620

CTTATGCATA TTCAGCGGTG AACTGAATAT TCATGAACGA GGCCCGTCCC GTCCCTCCCT 7680

CCTTCCCCCC ACCCCCGGAA CCCGCTCCGG AGGACCCGAA GGGCCCCGCC TTCATTACCG 7740

ATGCGTAGGA CAAACCATTT TCCCGATGTG TGTGGGGGGA TACTAATGAG AGACTTTAGC 7800

TGAAAAATGA GCCTGAACTC CGAAGCTGAG TAAAAATGGC CTAACTTTAT CCTCCGTTCT 7860

GTAAGTCCTC GGTTTGAGTG CACGGGAAAC CCGAAAGGAG GACGACAGGA CCAGGACATT 7920

CTCCTCCTCC TGTCGCGTCA GAAAGAACAC CCAACCAGGG AGCCGGAGCC CTAGCGTCAA 7980

CAACTCCGCC GCGCGCGCTC CGTGTAGGCC GGTGCGGGCG GCCCCGTAGC GCAAGGGAGG 8040

GCGGGAAAGG AAGGGGCGGG ACACAAGGGC GAATCTATAA AGGGCGTCAC TCAGCCAGTT 8100

CTCTCCTCAG AAGCGCCGAG AGCGCGACCG GGACGGTTGG AGAAGAAGGT GGCTCCCGGA 8160

AGGGGGAGAG ACAAACTGCC GTAACCTCTG CCGTTCAGGA TCATCGAATT CCTGCAGCCA 8220

ATATGGGATC GGCCATTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG 8280

AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT 8340

TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC 8400

TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT 8460

GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG 8520

TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG 8580

CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG 8640

CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG 8700

ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC 8760

GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA 8820

TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC 8880

GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG 8940

CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT 9000

ATCGCCTTCT TGACGAGTTC TTCTGAGGGG ATCAATTCTC TAGAGCTCGC TGATCAGCCT 9060

CGACTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA 9120

CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT 9180 GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG 9240

ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG 9300

AAAGAACCAG CTGGGGCTCG AGAGATCTTC ACAANGATAG GAAGGAGAGG AAGTGGGGCT 9360

CTGTTGATAG TTCTTGCTGA GCAGAAGCCN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 9960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10020

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNGG 10740

ATCCNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10800

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10860 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10920

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10980

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11040

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11100

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11160

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11220

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11280

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11340

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11400

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11460

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11520

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11580

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11640

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11700

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11760

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11820

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11880

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12060

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12120

NNNNNNNNNN NNNNNNNNNG GCGCCNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12540 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNC CATGGTCTAG AACTAGTGGA TCCCCCGGGC 12780

TGCAGGAATT CGATATCAAG CTTATCGATA CCGTCGACCT CGAGGGGGGG CCCGGTACCC 12840

AATTCGCCCT ATAGTGAGTC GTATTACGCG CGCTCACTGG CCGTCGTTTT ACAACGTCGT 12900

GACTGGGAAA ACCCTGGCGT TACCCAACTT AATCGCCTTG CAGCACATCC CCCTTTCGCC 12960

AGCTGGCGTA ATAGCGAAGA GGCCCGCACC GATCGCCCTT CCCAACAGTT GCGCAGCCTG 13020

AATGGCGAAT GGGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG 13080

CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT 13140

TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG GCTCCCTTTA 13200

GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT 13260

TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG 13320

TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT 13380

TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGCCTATT GGTTAAAAAA TGAGCTGATT 13440

TAACAAAAAT TTAACGCGAA TTTTAACAAA ATATTAACGC TTACAATTTA GGTGGCACTT 13500

TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT 13560

ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA 13620

TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG 13680

TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC TGAAGATCAG TTGGGTGCAC 13740

GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG 13800

AAGAACGTTT TCCAATGATG AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC 13860

GTATTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG 13920

TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT 13980

GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA CTTACTTCTG ACAACGATCG 14040

GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG GGATCATGTA ACTCGCCTTG 14100

ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC 14160

CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT 14220 CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT 14280

CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG AGCCGGTGAG CGTGGGTCTC 14340

GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC CCGTATCGTA GTTATCTACA 14400

CGACGGGGAG TCAGGCAACT ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT 14460

CACTGATTAA GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT 14520

TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA 14580

CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA 14640

AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC 14700

CACCGCTACC AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG 14760

TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTCCT TCTAGTGTAG CCGTAGTTAG 14820

GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC 14880

CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA AGACGATAGT 14940

TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG 15000

AGCGAACGAC CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC 15060

TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC 15120

GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC 15180

ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC CTATGGAAAA 15240

ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT 15300

TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCTTT GAGTGAGCTG 15360

ATACCGCTCG CCGCAGCCGA ACGACCGAGC GCAGCGAGTC AGTGAGCGAG GAAGCGGAAG 15420

AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC 15480

ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTGAGTTAGC 15540

TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA 15600

TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCGCG 15660

CAATTAACCC TCACTAAAGG GAACAAAAGC TG 15692

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15701 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: Swedish-FAD APP713

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 4807..4983

(ix) FEATURE:

(A) NAME/KEY: mutation

(B) LOCATION: replace (4835 , "")

(D) OTHER INFORMATION: /standard_name= "Swedish-FAD"

(ix) FEATURE:

(A) NAME/KEY: mutation

(B) LOCATION: replace (4981, "")

(D) OTHER INFORMATION: /standard_name= "APP713stop"

(ix) FEATURE:

(A) NAME/KEY: mat_j?eptide

(B) LOCATION: 8232..9032

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

GAGCTCCACC GCGGTGGCGG CCGCAAGTTT AAACATGGCG CGCCTTGTCG ACGGTATCGA 60

TAAGCTTGAT CTGATGGAAT TAGAACTTGG CAAAACAATA CTGAGAATGA AGTGTATGTG 120

GAACAGAGGC TGCTGATCTC GTTCTTCAGG CTATGAAACT GACACATTTG GAAACCACAG 180

TACTTAGAAC ACAAAGTGGG AATCAAGAGA AAAACAATGA TCCCACGAGA GATCTATAGA 240

TCTATAGATC ATGAGTGGAG GAATGAGCTG GCCCTTAATT TGGTTTTGCT TGTTTAAATT 300

ATGATATCCA ACTATGAAAC ATTATCATAA AGCAATAGTA AAGAGCCTTC AGTAAAGAGC 360

AGGCATTTAT CTAATCCCAC CCCACCCCCA CCCCCGTAGC TCCAATCCTT CCATTCAAAA 420

TGTAGGTACT CTGTTCTCAC CCTTCTTAAC AAAGTATGAC AGGAAAAAAC TTCCATTTTA 480

GTGGACATCT TTATTGTTTA ATAGATCATC AATTTCTGCA GGTCCTGGCC GGGGTCCCGC 540

GGAAACTCGG CCGTGGTGAC CAATACAAAA CAAAAGCGCT CCTCGTACCA GCGAAGAAGG 600

GGCAGAGATG CCGTAGTCAG GTTTAGTTCG TCCGGCGGCG CCAGAAATCC GCGCGGTGGT 660 TTTTGGGGGT CGGGGGTGTT TGGCAGCCAC AGACGCCCGG TGTTCGTGTC GCGCCAGTAC 720

ATGCGGTCCA TGCCCAGGCC ATCCAAAAAC CATGGGTCTG TCTGCTCAGT CCAGTCGTGG 780

ACCTGACCCC ACGCAACGCC CAAAAGAATA ACCCCCACGA ACCATAAACC ATTCCCCATG 840

GGGGACCCCG TCCCTAACCC ACGGGGCCCG TGGCTATGGC GGGCTTGCCG CCCCGACGTT 900

GGCTGCGAGC CCTGGGCCTT CACCCGAACT TGGGGGTTGG GGTGGGGAAA AGGAAGAAAC 960

GCGGGCGTAT TGGCCCCAAT GGGGTCTCGG TGGGGTATCG ACAGAGTGCC AGCCCTGGGA 1020

CCGAACCCCG CGTTTATGAA CAAACGACCC AACACCCGTG CGTTTTATTC TGTCTTTTTA 1080

TTGCCGTCAT AGCGCGGGTT CCTTCCGGTA TTGTCTCCTT CCGTGTTTCA GTTAGCCTCC 1140

CCCATCTCCC GGGCAAACGT GCGCGCCAGG TCGCAGATCG TCGGTATGGA GCCTGGGGTG 1200

GTGACGTGGG TCTGGACCAT CCCGGAGGTA AGTTGCAGCA GGGCGTCCCG GCAGCCGGCG 1260

GGCGATTGGT CGTAATCCAG GATAAAGACG TGCATGGGAC GGAGGCGTTT GGCCAAGACG 1320

TCCAAGGCCC AGGCAAACAC GTTATACAGG TCGCCGTTGG GGGCCAGCAA CTCGGGGGCC 1380

CGAAACAGGG TAAATAACGT GTCCCCGATA TGGGGTCGTG GGCCCGCGTT GCTCTGGGGC 1440

TCGGCACCCT GGGGCGGCAC GGCCGTCCCC GAAAGCTGTC CCCAATCCTC CCACCACGAC 1500

CCGCCGCCCT GCAGATACCG CACCGTATTG GCAAGCAGCC CGTAAACGCG GCGAATCGCG 1560

GCCAGCATAG CCAGGTCAAG CCGCTCGCCG GGGCGCTGGC GTTTGGCCAG GCGGTCGATG 1620

TGTCTGTCCT CCGGAAGGGC CCCCAACACG ATGTTTGTGC CGGGCAAGGT CGGCGGGATG 1680

AGGGCCACGA ACGCCAGCAC GGCCTGGGGG GTCATGCTGC CCATAAGGTA TCGCGCGGCC 1740

GGGTAGCACA GGAGGGCGGC GATGGGATGG CGGTCGAAGA TGAGGGTGAG GGCCGGGGGC 1800

GGGGCATGTG AACTCCCAGC CTCCCCCCCG ACATGAGGAG CCAGAACGGC GTCGGTCACG 1860

GCATAAGGCA TGCCCATTGT TATCTGGGCG CTTGTCATTA CCACCGCCGC GTCCCCGGCC 1920

GATATCTCAC CCTGGTCGAG GCGGTGTTGT GTGGTGTAGA TGTTCGCGAT TGTCTCGGAA 1980

GCCCCCAGCA CCTGCCAGTA AGTCATCGGC TCGGGTACGT AGACGATATC GTCGCGCGAA 2040

CCCAGGGCCA CCAGCAGTTG CGTGGTGGTG GTTTTCCCCA TCCCGTGAGG ACCGTCTATA 2100

TAAACCCGCA GTAGCGTGGG CATTTTCTGC TCCAGGCGGA CTTCCGTGGC TTCTTGCTGC 2160

CGGCGAGGGC GCAACGCCGT ACGTCGGTTG CTATGGCCGC GAGAACGCGC AGCCTGGTCG 2220

AACGCAGACG CGTGTTGATG GCAGGGGTAC GAAGCCATAC GCGCTTCTAC AAGGCGCTTG 2280

CCAAAGAGGT GCGGGAGTTT CACGCCACCA AGATCTGCGG CACGCTGTTG ACGCTGTTAA 2340 GCGGGTCGCT GCAGGTCGAA AGGCCCGGAG ATGAGGAAGA GGAGAACAGC GCGGCAGACG 2400

TGCGCTTTTG AAGCGTGCAG AATGCCGGGC TCCGGAGGAC CTTCGCGCCC GCCCCGCCCC 2460

TGAGCCCGCC CCTGAGCCCG CCCCCGGACC CACCCCTTCC CAGCCTCTGA GCCCAGAAAG 2520

CGAAGGAGCA AAGCTGCTAT TGGCCGCTGC CCCAAAGGCC TACCCGCTTC CATTGCTCAG 2580

CGGTGCTGTC CATCTGCACG AGACTAGTGA GACGTGCTAC TTCCATTTGT CACGTCCTGC 2640

ACGACGCGAG CTGCGGGGCG GGGGGGAACT TCCTGACTAG GGGAGGAGTA GAAGGTGGCG 2700

CGAAGGGGCC ACCAAAGAAG GGAGCCGGTT GGCGCTACCG GTGGATGTGG AATGTGTGCG 2760

AGGCCAGAGG CCACTTGTGT AGCGCCAAGT GCCAGCGGGG CTGCTAAAGC GCATGCTCCA 2820

GACTGCCTTG GGAAAAGCGC CTCCCCTACC CCGGTAGAAT TCCTGCAGCC CGGGGGATCC 2880

ACTAGTTCTA GAGCGGCCGC TCTGACCATG GNNNNNNNNN NNNNNNNNNA AGCTTNNNNN 2940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3060

NNNNNNNNNN NNNNNNNCAT ATGNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3420

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNGAATTCN NNNNNNNNNN 3600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3780

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNGAATT CNNNNNNNNN 3840

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3900

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3960

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNrøNNNNN NNNNNNNNNN NNNNNNNNNN 4020 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4080

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNG AATTCNNNNN 4140

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4200

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4260

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4320

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4380

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4440

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4500

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4560

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4620

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4680

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4740

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 4800

NNNNGTTCTG GGCTGACAAA CATCAAGACG GAAGAGATCT CTGAAGTGAA TCTAGATGCA 4860

GAATTCCGAC ATGACTCAGG ATATGAAGTT CATCATCAAA AATTGGTGTT CTTTGCAGAA 4920

GATGTGGGTT CAAACAAAGG TGCAATCATT GGACTCATGG TGGGCGGTGT TGTCATAGCG 4980

TAGGGATCCA CAGTGATCGT CATCACCTTG GTGATGCTGA AGAAGAAACA GTACACATCC 5040

ATTCATCATG GTGTGGTGGA GGTTGACGCC GCTGTCACCC CAGAGGAGCG CCACCTGTCC 5100

AAGATGCAGC AGAACGGCTA CGAAAATCCA ACCTACAAGT TCTTTGAGCA GATGCAGAAC 5160

TAGACCCCCG CCACAGCAGC CTCTGAAGTT GGACAGCAAA ACCATTGCTT CACTACCCAT 5220

CGGTGTCCAT TTATAGAATA ATGTGGGAAG AAACAAACCC GTTTTATGAT TTACTCATTA 5280

TCGCCTTTTG ACAGCTGTGC TGTAACACAA GTAGATGCCT GAACTTGAAT TAATCCACAC 5340

ATCAGTAATG TATTCTATCT CTCTTTACAT TTTGGTCTCT ATACTACATT ATTAATGGGT 5400

TTTGTGTACT GTAAAGAATT TAGCTGTATC AAACTAGTGC ATGAATAGAT TCTCTCCTGA 5460

TTATTTATCA CATAGCCCCT TAGCCAGTTG TATATTATTC TTGTGGTTTG TGACCCAATT 5520

AAGTCCTACT TTACATATGC TTTAAGAATC GATGGGGGAT GCTTCATGTG AACGTGGGAG 5580

TTCAGCTGCT TCTCTTGCCT AAGTATTCCT TTCCTGATCA CTATGCATTT TAAAGTTAAA 5640

CATTTTTAAG TATTTCAGAT GCTTTAGAGA GATTTTTTTT CCATGACTGC ATTTTACTGT 5700 ACAGATTGCT GCTTCTGCTA TATTTGTGAT ATAGGAATTA AGAGGATACA CACGTTTGTT 5760

TCTTCGTGCC TGTTTTATGT GCACACATTA GGCATTGAGA CTTCAAGCTT TTCTTTTTTT 5820

GTCCACGTAT CTTTGGGTCT TTGATAAAGA AAAGAATCCC TGTTCATTGT AAGCACTTTT 5880

ACGGGGCGGG TGGGGAGGGG TGCTCTGCTG GTCTTCAATT ACCAAGAATT CTCCAAAACA 5940

ATTTTCTGCA GGATGATTGT ACAGAATCAT TGCTTATGAC ATGATCGCTT TCTACACTGT 6000

ATTACATAAA TAAATTAAAT AAAATAACCC CGGGCAAGAC TTTTCTTTGA AGGATGACTA 6060

CAGACATTAA ATAATCGAAG TAATTTTGGG TGGGGAGAAG AGGCAGATTC AATTTTCTTT 6120

AACCAGTCTG AAGTTTCATT TATGATACAA AAGAAGATGA AAATGGAAGT GGCAATATAA 6180

GGGGATGAGG AAGGCATGCC TGGACAAACC CTTCTTTTAA GATGTGTCTT CAATTTGTAT 6240

AAAATGGTGT TTTCATGTAA ATAAATACAT TCTTGGAGGA GCCACATTGT GCTGGTGTGA 6300

ATGATTCCAT AGTAACAATC TTGACCATTT ACTGACGTAC AGACCAGTGA GAAGTCTTCG 6360

CATGTTGGGT ACCCACACCT GTTGTGTCTT AATTGCAAGT CTGAGTAGGA AGTTGGGGCC 6420

AACATGTGTC TCCCAGTGCT GGGAAAATAT TTCATAGACC TAATTTACAG TCTTTACTTG 6480

ATCTAAAACA TTTTGCTGCC ATATTTTGGC CCTCAAGTTT GTCCCAAATG AGAGACAAAG 6540

GGAAAAGTTC CAGGGAAATA AAAATTAAGA CAGCTGATTA TCTGTAAAGC ATGGTTTCTC 6600

ATCCTGAACG CTACTAACAT TTTGCAGGGA ATAATTCCTT GTTGAAGGGA GTTGTCCTGA 6660

CCAGTGTAGG ATATTTATTT ATTTTATTTA TGTTTTTTGA GACGGAGTCT CGCTCTGTCA 6720

CCCAGGCTGG AGTGCAGTGG CACAATCTCG GCTCACTGCA AGCTCCGCCT CCCGGGTTCA 6780

CGCCATTCTC CTGCCTCAGC CTCCTGAATA GCTGGGACTC TAGGTGCCCG CCACCACGCC 6840

CGGCTAATTT TTTGTATTTT TAGTAGAGAC GGGGTTTCAC CGTGTTAGCC AGGACAGTCT 6900

TGGTCTCCTG ACCTCGTGAT CTGCCTGCCT CGGCCTCCCA AAGTGCTGAG ATTACAGGCG 6960

TGCAAGCCGC GCCCAGCCAG TGCTCTCCTT TTAAAAGTAG CCCATTGGCT GGGCGCAGTG 7020

GCTCACGCCT GTAATCCCAG CACTTTGGGA GGCTGAGGCG GGTGGATCAC GAGGTCAGGA 7080

GATCAAGAAT ATCCTGGCCA ATATGGTGAA ACCCCATCTC TACTAAAAAT ACAAAAAAAA 7140

AAAAAAAAAA AAAAAGGCCG GGCATGGTGG CGGGCGCTTG TAGTCCCAGC TACTCAGGAG 7200

GCTGAGGCAG GAGAATGGTG TGCACCTGGG AGGCGGAGGT TGCAGTGAGC TGAGATCGCG 7260

CCACTGCACT CCAGCCTGGG AGACAGAGCG AGACTCCGTC TCAATAAATA AATAAATAAA 7320

TAAATAAAAG GAGGGCCTGG CACGAATGAC ATGCAGGGAA GGCAGTGAGC AGGTGGAGGT 7380 CCCTGTACTC GTTGTGGTGC CTTATCTACC AGGCGGTTGA GTTGACGTCT TTGTGGACAG 7440

AATTCGAGCT CGGTACCCGG GGATCCTCTA GAGTCGACCT TAAGGTCGAC GGTATCGATA 7500

AGCTTGGGCT TGAACATCGA GCGCCAGGGC TCCGTAAAGC TACTAGAGCA CAGGCGGTGC 7560

CCCAACGTCC TGGGGCCTCT CCACTAATAA CGGCTACTTC CAATTGATTG GACGCGCCAT 7620

CTTGCCTGCC TTATGCATAT TCAGCGGTGA ACTGAATATT CATGAACGAG GCCCGTCCCG 7680

TCCCTCCCTC CTTCCCCCCA CCCCCGGAAC CCGCTCCGGA GGACCCGAAG GGCCCCGCCT 7740

TCATTACCGA TGCGTAGGAC AAACCATTTT CCCGATGTGT GTGGGGGGAT ACTAATGAGA 7800

GACTTTAGCT GAAAAATGAG CCTGAACTCC GAAGCTGAGT AAAAATGGCC TAACTTTATC 7860

CTCCGTTCTG TAAGTCCTCG GTTTGAGTGC ACGGGAAACC CGAAAGGAGG ACGACAGGAC 7920

CAGGACATTC TCCTCCTCCT GTCGCGTCAG AAAGAACACC CAACCAGGGA GCCGGAGCCC 7980

TAGCGTCAAC AACTCCGCCG CGCGCGCTCC GTGTAGGCCG GTGCGGGCGG CCCCGTAGCG 8040

CAAGGGAGGG CGGGAAAGGA AGGGGCGGGA CACAAGGGCG AATCTATAAA GGGCGTCACT 8100

CAGCCAGTTC TCTCCTCAGA AGCGCCGAGA GCGCGACCGG GACGGTTGGA GAAGAAGGTG 8160

GCTCCCGGAA GGGGGAGAGA CAAACTGCCG TAACCTCTGC CGTTCAGGAT CATCGAATTC 8220

CTGCAGCCAA TATGGGATCG GCCATTGAAC AAGATGGATT GCACGCAGGT TCTCCGGCCG 8280

CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC TGCTCTGATG 8340

CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG ACCGACCTGT 8400

CCGGTGCCCT GAATGAACTG CAGGACGAGG CAGCGCGGCT ATCGTGGCTG GCCACGACGG 8460

GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC TGGCTGCTAT 8520

TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC GAGAAAGTAT 8580

CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG 8640

ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC GGTCTTGTCG 8700

ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG TTCGCCAGGC 8760

TCAAGGCGCG CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT GCCTGCTTGC 8820

CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT CGACTGTGGC CGGCTGGGTG 8880

TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA GAGCTTGGCG 8940

GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT TCGCAGCGCA 9000

TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGGGGA TCAATTCTCT AGAGCTCGCT 9060 OfrΛOT NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

08901 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

02901 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

09S0I NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

OOSOI NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

OfrfrOI NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

08ε0T NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN oεεoτ NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

09Z0I NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

00Z0I NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

OfrTOI NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

08001 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

OSOOt NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0966 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0066 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0fr86 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

08Z.6 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0 ZL6 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0996 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0096 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

0frS6 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

08fr6 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN

03fr6 NNNNNNNNNN NNNNNNNNNN NNDDOWOVO 9V9XD9XXDX XOVXV9XX9X DXDD990X0V

09€6 V99VDV9DW G0VXV9NWD VDXXDXVDVO V9DXD099GX DDVDDW9W V99D99VDX0

00 6 XXDDOXVXDX D999X9909X V9D99XD9XV 099VD9VXW DV9W099XX V09V99099V

0frZ6 YD0YDY99YD 9990X9999X D999DGXDXX VXDXXVDX9X 9DVX9V9XDX 0XXVD90XVD

0816 GXXVW99V9 XWWXWXD DXXXDDXOXD YDD9XDYDD9 X99W99XDD DV9XXDDXXD

0εi6 D9X9DDDD0X 9DDD9XXX9X XOXDXVDDOV DD9XX9YXDX XDD9X9XDY9 DXD09VDXVG

- oετ -

LOSP VLβSn/lDd 0SI60/66 OΛV NNNNNNNGGA TCCNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10800

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10860

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10920

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 10980

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11040

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11100

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11160

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11220

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11280

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11340

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11400

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11460

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11520

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11580

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11640

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11700

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11760

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11820

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11880

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 11940

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12000

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12060

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12120

NNNNNNNNNN NNNNNNNNNN NNNNNNNNGG CGCCNNNNNN NNNNNNNNNN NNNNNNNNNN 12180

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12240

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12300

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12360

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12420 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12480

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12540

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12600

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12660

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 12720

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNCC ATGGTCTAGA ACTAGTGGAT 12780

CCCCCGGGCT GCAGGAATTC GATATCAAGC TTATCGATAC CGTCGACCTC GAGGGGGGGC 12840

CCGGTACCCA ATTCGCCCTA TAGTGAGTCG TATTACGCGC GCTCACTGGC CGTCGTTTTA 12900

CAACGTCGTG ACTGGGAAAA CCCTGGCGTT ACCCAACTTA ATCGCCTTGC AGCACATCCC 12960

CCTTTCGCCA GCTGGCGTAA TAGCGAAGAG GCCCGCACCG ATCGCCCTTC CCAACAGTTG 13020

CGCAGCCTGA ATGGCGAATG GGACGCGCCC TGTAGCGGCG CATTAAGCGC GGCGGGTGTG 13080

GTGGTTACGC GCAGCGTGAC CGCTACACTT GCCAGCGCCC TAGCGCCCGC TCCTTTCGCT 13140

TTCTTCCCTT CCTTTCTCGC CACGTTCGCC GGCTTTCCCC GTCAAGCTCT AAATCGGGGG 13200

CTCCCTTTAG GGTTCCGATT TAGTGCTTTA CGGCACCTCG ACCCCAAAAA ACTTGATTAG 13260

GGTGATGGTT CACGTAGTGG GCCATCGCCC TGATAGACGG TTTTTCGCCC TTTGACGTTG 13320

GAGTCCACGT TCTTTAATAG TGGACTCTTG TTCCAAACTG GAACAACACT CAACCCTATC 13380

TCGGTCTATT CTTTTGATTT ATAAGGGATT TTGCCGATTT CGGCCTATTG GTTAAAAAAT 13440

GAGCTGATTT AACAAAAATT TAACGCGAAT TTTAACAAAA TATTAACGCT TACAATTTAG 13500

GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 13560

CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 13620

GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT 13680

GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 13740

TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 13800

TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 13860

TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 13920

ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 13980

GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 14040

CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA 14100 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 14160

CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 14220

CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 14280

TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 14340

GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 14400

TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 14460

TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 14520

AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 14580

ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG 14640

AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 14700

CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 14760

TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 14820

CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 14880

TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 14940

GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 15000

CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 15060

GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 15120

CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 15180

GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 15240

TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG 15300

CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG 15360

AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 15420

AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 15480

GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG 15540

TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 15600

TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG 15660

CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT G 15701

(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1297 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

GCATGCCTGG ACAAACCCTT CTTTTAAGAT GTGTCTTCAA TTTGTATAAA ATGGTGTTTT 60

CATGTAAATA AATACATTCT TGGAGGAGCC ACATTGTGCT GGTGTGAATG ATTCCATAGT 120

AACAATCTTG ACCATTTACT GACGTACAGA CCAGTGAGAA GTCTTCGCAT GTTGGGTACC 180

CACACCTGTT GTGTCTTAAT TGCAAGTCTG AGTAGGAAGT TGGGGCCAAC ATGTGTCTCC 240

CAGTGCTGGG AAAATATTTC ATAGACCTAA TTTACAGTCT TTACTTGATC TAAAACATTT 300

TGCTGCCATA TTTTGGCCCT CAAGTTTGTC CCAAATGAGA GACAAAGGGA AAAGTTCCAG 360

GGAAATAAAA ATTAAGACAG CTGATTATCT GTAAAGCATG GTTTCTCATC CTGAACGCTA 420

CTAACATTTT GCAGGGAATA ATTCCTTGTT GAAGGGAGTT GTCCTGACCA GTGTAGGATA 480

TTTATTTATT TTATTTATGT TTTTTGAGAC GGAGTCTCGC TCTGTCACCC AGGCTGGAGT 540

GCAGTGGCAC AATCTCGGCT CACTGCAAGC TCCGCCTCCC GGGTTCACGC CATTCTCCTG 600

CCTCAGCCTC CTGAATAGCT GGGACTCTAG GTGCCCGCCA CCACGCCCGG CTAATTTTTT 660

GTATTTTTAG TAGAGACGGG GTTTCACCGT GTTAGCCAGG ACAGTCTTGG TCTCCTGACC 720

TCGTGATCTG CCTGCCTCGG CCTCCCAAAG TGCTGAGATT ACAGGCGTGC AAGCCGCGCC 780

CAGCCAGTGC TCTCCTTTTA AAAGTAGCCC ATTGGCTGGG CGCAGTGGCT CACGCCTGTA 840

ATCCCAGCAC TTTGGGAGGC TGAGGCGGGT GGATCACGAG GTCAGGAGAT CAAGAATATC 900

CTGGCCAATA TGGTGAAACC CCATCTCTAC TAAAAATACA AAAAAAAAAA AAAAAAAAAA 960

AAGGCCGGGC ATGGTGGCGG GCGCTTGTAG TCCCAGCTAC TCAGGAGGCT GAGGCAGGAG 1020

AATGGTGTGC ACCTGGGAGG CGGAGGTTGC AGTGAGCTGA GATCGCGCCA CTGCACTCCA 1080

GCCTGGGAGA CAGAGCGAGA CTCCGTCTCA ATAAATAAAT AAATAAATAA ATAAAAGGAG 1140

GGCCTGGCAC GAATGACATG CAGGGAAGGC AGTGAGCAGG TGGAGGTCCC TGTACTCGTT 1200 GTGGTGCCTT ATCTACCAGG CGGTTGAGTT GACGTCTTTG TGGACAGAAT TCGAGCTCGG 1260 TACCCGGGGA TCCTCTAGAG TCGACCTGCA GGCATGC 1297

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

CCATCGATGG ATCAGTTACG GAAACGATGC TCTCATGC 38

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Primer"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

CCATCGATGG CCAAGGTGAT GACGATCACT GTGGATCCCT ACGCTATGAC AACACCGC 58

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 348 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: Swedish-FAD APP

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..348

(D) OTHER INFORMATION: /standard_name= "Swedish-FAD APP"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

TCT GGG CTG ACA AAC ATC AAG ACG GAA GAG ATC TCT GAA GTG AAT CTA 48

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Asn Leu 1 5 10 15

GAT GCA GAA TTC CGA CAT GAC TCA GGA TAT GAA GTT CAT CAT CAA AAA 96 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 20 25 30

TTG GTG TTC TTT GCA GAA GAT GTG GGT TCA AAC AAA GGT GCA ATC ATT 144 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 35 40 45

GGA CTC ATG GTG GGC GGT GTT GTC ATA GCG ACA GTG ATC GTC ATC ACC 192 Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr 50 55 60

TTG GTG ATG CTG AAG AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG 240 Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val 65 70 75 80

GTG GAG GTT GAC GCC GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG 288 Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys 85 90 95

ATG CAG CAG AAC GGC TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG 336 Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin 100 105 110

ATG CAG AAC TAG 348

Met Gin Asn *

(2) INFORMATION FOR SEQ ID NO: 26

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 115 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Asn Leu 1 5 10 15 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 20 25 30

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 35 40 45

Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr 50 55 60

Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val 65 70 75 80

Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys 85 90 95

Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin 100 105 110

Met Gin Asn 115

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 804 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..804

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

ATG GGA TCG GCC ATT GAA CAA GAT GGA TTG CAC GCA GGT TCT CCG GCC 48 Met Gly Ser Ala He Glu Gin Asp Gly Leu His Ala Gly Ser Pro Ala 1 5 10 15

GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC TGG GCA CAA CAG ACA ATC 96 Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gin Gin Thr He 20 25 30

GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG TCA GCG CAG GGG CGC CCG 144 Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gin Gly Arg Pro 35 40 45

GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC CTG AAT GAA CTG CAG 192 Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gin 50 55 60

GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC ACG ACG GGC GTT CCT TGC 240 Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys 65 70 75 80

GCA GCT GTG CTC GAC GTT GTC ACT GAA GCG GGA AGG GAC TGG CTG CTA 288 Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu 85 90 95

TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA TCT CAC CTT GCT CCT 336 Leu Gly Glu Val Pro Gly Gin Asp Leu Leu Ser Ser His Leu Ala Pro 100 105 110

GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG CGG CGG CTG CAT ACG 384 Ala Glu Lys Val Ser He Met Ala Asp Ala Met Arg Arg Leu His Thr 115 120 125

CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA GCG AAA CAT CGC ATC 432 Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gin Ala Lys His Arg He 130 135 140

GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT GTC GAT CAG GAT GAT 480 Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gin Asp Asp 145 150 155 160

CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC GAA CTG TTC GCC AGG 528 Leu Asp Glu Glu His Gin Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg 165 170 175

CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC GTC GTG ACC CAT GGC 576 Leu Lys Ala Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly 180 185 190

GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT GGC CGC TTT TCT GGA 624 Asp Ala Cys Leu Pro Asn He Met Val Glu Asn Gly Arg Phe Ser Gly 195 200 205

TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC CGC TAT CAG GAC ATA 672 Phe He Asp Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gin Asp He 210 215 220

GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT GGC GGC GAA TGG GCT 720 Ala Leu Ala Thr Arg Asp He Ala Glu Glu Leu Gly Gly Glu Trp Ala 225 230 235 240

GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT CCC GAT TCG CAG CGC 768 Asp Arg Phe Leu Val Leu Tyr Gly He Ala Ala Pro Asp Ser Gin Arg 245 250 255

ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC TTC TAG 804

He Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe * 260 265

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 267 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Met Gly Ser Ala He Glu Gin Asp Gly Leu His Ala Gly Ser Pro Ala 1 5 10 15

Ala Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gin Gin Thr He 20 25 30

Gly Cys Ser Asp Ala Ala Val Phe Arg Leu Ser Ala Gin Gly Arg Pro 35 40 45

Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gin 50 55 60

Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys 65 70 75 80

Ala Ala Val Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu 85 90 95

Leu Gly Glu Val Pro Gly Gin Asp Leu Leu Ser Ser His Leu Ala Pro 100 105 110

Ala Glu Lys Val Ser He Met Ala Asp Ala Met Arg Arg Leu His Thr 115 120 125 Leu Asp Pro Ala Thr Cys Pro Phe Asp His Gin Ala Lys His Arg He 130 135 140

Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gin Asp Asp 145 150 155 160

Leu Asp Glu Glu His Gin Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg 165 170 175

Leu Lys Ala Arg Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly 180 185 190

Asp Ala Cys Leu Pro Asn He Met Val Glu Asn Gly Arg Phe Ser Gly 195 200 205

Phe He Asp Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gin Asp He 210 215 220

Ala Leu Ala Thr Arg Asp He Ala Glu Glu Leu Gly Gly Glu Trp Ala 225 230 235 240

Asp Arg Phe Leu Val Leu Tyr Gly He Ala Ala Pro Asp Ser Gin Arg 245 250 255

He Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe 260 265

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 348 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: London-FAD APP

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..348

(D) OTHER INFORMATION: /standard_name= "London-FAD APP"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TCT GGG CTG ACA AAC ATC AAG ACG GAA GAG ATC TCT GAA GTG AAG ATG 48

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met 1 5 10 15

GGA CTC ATG GTG GGC GGT GTT GTC ATA GCG ACA GTG ATA ATC ATC ACC 192 Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He He He Thr 50 55 60

ATG CAG AAC TAG 348

Met Gin Asn *

(2) INFORMATION FOR SEQ ID NO: 30

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 115 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met 1 5 10 15

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 20 25 30

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 35 40 45

Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He He He Thr 50 55 60

Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val 65 70 75 80

Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys 85 90 95

Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin 100 105 110

Met Gin Asn 115

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 348 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: Swedish-FAD APP

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: London-FAD APP

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..348

(D) OTHER INFORMATION: /standard_name= "Swedish-FAD/London-FAD"

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

TCT GGG CTG ACA AAC ATC AAG ACG GAA GAG ATC TCT GAA GTG AAT CTA 48

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Asn Leu 1 5 10 15

ATG CAG AAC TAG 348

Met Gin Asn *

(2) INFORMATION FOR SEQ ID NO: 32

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 115 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Asn Leu 1 5 10 15

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 20 25 30

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 35 40 45

Gly Leu Met Val Gly Gly Val Val He Ala Thr Val He He He Thr 50 55 60

Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val 65 70 75 80

Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys 85 90 95

Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin 100 105 110

Met Gin Asn 115

(2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 177 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Targetting vector"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..177

(D) OTHER INFORMATION: /standard_name= "APP713stop"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

TCT GGG CTG ACA AAC ATC AAG ACG GAA GAG ATC TCT GAA GTG AAT CTA 48

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Asn Leu 1 5 10 15

GGA CTC ATG GTG GGC GGT GTT GTC ATA GCG TAG 177

Gly Leu Met Val Gly Gly Val Val He Ala * 50 55

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Asn Leu 1 5 10 15

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 20 25 30

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He 35 40 45 Gly Leu Met Val Gly Gly Val Val He Ala 50 55

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 99 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

TCT GGG CTG ACA AAC ATC AAG ACG GAA GAG ATC TCT GAA GTG AAG ATG 48

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met 1 5 10 15

CTG 99

Leu

(2) INFORMATION FOR SEQ ID NO: 36

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met 1 5 10 15

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 20 25 30

Leu

Claims

What is claimed:

1. A recombinant nucleic acid molecule comprising: a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a rodent APP gene, which is operably linked to the 5 ' terminus of a nucleotide coding sequence which, when inserted into said APP gene, codes for at least one amino acid whose identity and/or position is not naturally-occurring in said APP gene, and a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a rodent APP gene, which is operably linked to the 3' terminus of said nucleotide coding sequence.

2. A recombinant nucleic acid molecule of claim 1 , wherein said nucleotide coding sequence comprises at least one amino acid coded for by a human APP gene.

3. A recombinant nucleic acid molecule of claim 1, wherein said nucleotide coding sequence codes without interruption for an amino acid sequence, which amino acid sequence is coded for by two or more exons in a naturally-occurring genomic sequence.

4. A recombinant nucleic acid molecule of claim 1, wherein said nucleotide coding sequence codes without interruption for an amino acid sequence of a human APP polypeptide, which amino acid sequence is coded for by two or more exons in a human APP genomic sequence.

5. A recombinant nucleic acid molecule of claim 1, wherein said nucleotide coding sequence hybridizes under stringent conditions to a nucleotide sequence coding without interruption for at least one amino acid sequence coded for by a human APP gene.

6. A recombinant nucleic acid molecule of claim 1, wherein said nucleotide coding sequence, which when inserted into a rodent APP gene, comprises an asparagine at amino acid position 670, a leucine at amino acid position 671, an arginine at amino acid position 676, a threonine at amino acid position 681, a histidine at amino acid 684, an isoleucine at amino acid 717, or a combination thereof.

7. A recombinant nucleic acid molecule of claim 1, wherein said nucleotide coding sequence, which when inserted into a rodent APP gene, does not comprise an asparagine at amino acid position 670, a leucine at amino acid position 671, an arginine at amino acid position 676, a threonine at amino acid position 681, and a histidine at amino acid 684.

8. A recombinant nucleic acid molecule of claim 1, further comprising a selectable marker gene.

9. A recombinant nucleic acid molecule of claim 8, wherein the selectable marker gene codes for neomycin resistance.

10. A recombinant nucleic acid molecule of claim 1 , further comprising a vector.

11. A recombinant nucleic acid molecule of claim 10, further comprising a selectable marker gene, wherein said vector contains only one selectable marker gene which can be selected for in a mammalian host.

12. A recombinant nucleic acid molecule of claim 1 , further comprising a selectable marker gene and a vector, and wherein said nucleotide coding sequence codes without interruption for an amino acid sequence, which amino acid sequence is coded for by two or more exons in a human APP genomic sequence.

13. A recombinant nucleic acid molecule of claim 1, wherein the nucleotide coding sequence is at least part of a cDNA.

14. A recombinant nucleic acid molecule of claim 1 , wherein said nucleotide sequence operably linked to the 5 ' terminus of said nucleotide coding sequence comprises all or part of intron 15 of a mouse APP gene.

15. A recombinant nucleic acid molecule of claim 1 , wherein said nucleotide sequence operably linked to the 3 ' terminus of said nucleotide coding sequence comprises all or part of intron 16 of a mouse APP gene.

16. A recombinant nucleic acid molecule comprising: a nucleotide sequence of a rodent APP gene which is operably linked to the 5' terminus of a nucleotide coding sequence which, when inserted into a mouse APP gene, codes for at least one amino acid whose identity and/or position is not naturally-occurring in said APP gene, and a nucleotide sequence of a rodent APP gene which is operably linked to the 3' terminus of said nucleotide coding sequence coding, whereby the nucleic acid molecule is effective to achieve homologous recombination in a rodent chromosome.

17. A recombinant nucleic acid coding for a humanized rodent APP polypeptide comprising at least one amino acid coded for by a human APP gene.

18. A recombinant nucleic acid of claim 17 which codes without interruption for said humanized rodent APP polypeptide, and which comprises two or more amino acids coded for by a human APP gene, wherein said amino acids of the human APP gene are coded for by two or more exons in a human APP genomic sequence.

19. A recombinant nucleic acid of claim 18, wherein the rodent is a mouse, comprising an asparagine at amino acid position 670, a leucine at amino acid position 671, an arginine at amino acid position 676, a threonine at amino acid position 681, a histidine at amino acid 684, an isoleucine at amino acid 717, or a combination thereof.

20. A humanized mouse APP polypeptide coded for by a recombinant nucleic acid of claim 19.

21. A transformed non-human mammal cell comprising a recombinant nucleic acid of claim 1.

22. A transformed mouse cell comprising a recombinant nucleic acid of claim 1.

23. A transformed mouse cell of claim 22, wherein the mouse cell is an embryonic stem cell.

24. A transformed mouse cell comprising a recombinant nucleic acid of claim 1 , wherein the nucleic acid is integrated into the chromosome of the mouse cell at the mouse APP gene locus.

25. A transformed mouse cell comprising a recombinant nucleic acid of claim 4.

26. A transformed mouse cell comprising a recombinant nucleic acid of claim 5.

27. A transformed mouse cell comprising a recombinant nucleic acid of claim 6.

28. A transformed mouse cell comprising a recombinant nucleic acid of claim 12.

29. A transgenic rodent comprising cells which contain a recombinant APP gene integrated into a chromosome of said cell at the APP gene locus, said APP gene comprising a nucleotide coding sequence which codes for a recombinant APP polypeptide comprising at least one amino acid whose identity and/or position is not naturally-occurring in said rodent APP gene.

30. A transgenic rodent comprising cells which contain a recombinant APP gene comprising a nucleotide coding sequence for a recombinant APP polypeptide, said APP polypeptide comprising two or more amino acids coded for by a human APP gene, wherein said amino acids of the human APP gene are coded for by two or more exons in a human APP genomic sequence.

31. A transgenic rodent of claim 30 which is a mouse, wherein said APP polypeptide comprises an asparagine at amino acid position 670, a leucine at amino acid position 671, an arginine at amino acid position 676, a threonine at amino acid position 681, a histidine at amino acid 684, an isoleucine at amino acid 717, or a combination thereof.

32. A transgenic rodent of claim 30, wherein said recombinant APP gene further comprises a selectable marker gene.

33. A transgenic mouse of claim 32, wherein said selectable marker gene codes for neomycin resistance.

34. A transgenic mouse of claim 30, wherein said recombinant APP gene is integrated into the rodent chromosome at the APP gene locus.

35. A transgenic mouse of claim 30, wherein the recombinant APP gene is expressed in the brain of said mouse in an amount effective to produce neuronal cell degeneration and/or apoptosis.

36. A transgenic mouse of claim 30, wherein the recombinant APP gene is expressed in the brain of said mouse in an amount effective to cause a behavioral or cognitive dysfunction, wherein the dysfunction is conferred by said polypeptide coded for by said recombinant APP gene.

37. A transgenic mouse of claim 29, wherein the recombinant APP gene is expressed in the brain of said rodent in an amount effective to produce neuronal cell degeneration and/or apoptosis.

38. A transgenic rodent of claim 29, wherein the recombinant APP gene is expressed in the brain of said rodent in an amount effective to cause a behavioral or cognitive dysfunction, wherein the dysfunction is conferred by said polypeptide coded for by said recombinant APP gene.

39. A method for producing a transgenic rodent comprising a recombinant nucleic acid molecule, comprising:

(a) introducing a nucleic acid molecule of claim 1 into mouse ES cells;

(b) culturing said ES cells under conditions effective for homologous recombination between said nucleic acid and an APP gene of said ES cells;

(c) selecting cells having a nucleic acid of claim 1 integrated by homologous recombination into said APP gene of said ES cells;

(d) introducing said transformed ES cells into a blastocyst;

(e) implanting said blastocyst into a pseudopregnant rodent; and

(f) allowing said embryo to develop to term.

40. A method of claim 39, wherein only one APP gene targeting event of steps (a)-(c) is accomplished.

41. A transgenic rodent produced according to claim 39.

42. A method of claim 39, further comprising identifying at least one transgenic offspring containing said recombinant DNA.

43. A method of claim 39, further comprising breeding said offspring to form a transgenic line of rodents.

44. A method of claim 39, wherein said nucleotide coding sequence codes without interruption for an amino acid sequence, and wherein the amino acid sequence is coded for by two or more exons in a human APP genomic sequence.

45. A method for producing a transgenic rodent having a phenotype mediated by expression of a recombinant APP gene in the brain of said rodent, wherein the APP gene is expressed in the brain of said rodent in an amount effective to produce neuronal cell degeneration and/or apoptosis and/or in an amount effective to cause a behavioral or cognitive dysfunction, said method comprising:

(a) introducing a nucleic acid molecule of claim 1 into rodent ES cells;

(c) selecting cells having a nucleic acid of claim 1 integrated into

(d) introducing said transformed ES cells into a blastocyst;

(e) implanting said blastocyst into a pseudopregnant rodent; and

(f) allowing said embryo to develop to term.

46. A method of claim 45, wherein said nucleotide coding sequence codes without interruption for an amino acid sequence, wherein the amino acid sequence is coded for by two or more exons in a human APP genomic sequence.

47. A method of screening a compound for an effect on a phenotype mediated by expression of a recombinant APP gene in the brain of a rodent, comprising: administering said compound to a rodent of claim 30 expressing said recombinant APP gene, and observing whether an effect on said phenotype results.

48. A recombinant nucleic acid molecule comprising: a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a target gene, operably linked to the 5' terminus of a nucleotide coding sequence which codes without interruption for an amino acid sequence, which when inserted into said target gene, codes for at least two amino acids whose identity and/or position is not naturally- occurring in said target gene, wherein said amino acids are coded for by two or more exons in a genomic sequence, and a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a non-human gene, operably linked to the 3' terminus of said nucleotide coding sequence.

49. A recombinant nucleic acid of claim 48, wherein the non-human mammal is a mouse.

50. A method for producing a transgenic rodent comprising a recombinant nucleic acid molecule, comprising:

(a) introducing a nucleic acid molecule of claim 48 into rodent ES cells;

(b) culturing said ES cells under conditions effective for homologous recombination between said nucleic acid and a target gene of said ES cells; (c) selecting cells having a nucleic acid of claim 48 integrated by homologous recombination into said target gene of said ES cells;

(d) introducing said transformed ES cells into a blastocyst;

(e) implanting said blastocyst into a pseudopregnant mouse; and

(f) allowing said embryo to develop to term.

51. A method of claim 50, wherein steps (a)-(c) are accomplished only once.

52. A recombinant nucleic acid molecule comprising: a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a target gene, operably linked to the 5' terminus of a nucleotide coding sequence which codes without interruption for an amino acid sequence, which when inserted into said target gene, codes for at least one amino acid whose identity and/or position is not naturally- occurring in said target gene, and a nucleotide sequence which is effective to achieve homologous recombination at a predefined position of a target gene, operably linked to the 3' terminus of said nucleotide coding sequence.

53. A nucleic acid molecule of claim 52, wherein the target gene is a mammalian gene.

54. A method for producing a transgenic rodent comprising a recombinant nucleic acid molecule, comprising:

(a) introducing a nucleic acid molecule of claim 52 into rodent ES cells; (b) culturing said ES cells under conditions effective for homologous recombination between said nucleic acid and a target gene of said ES cells;

(c) selecting cells having a nucleic acid of claim 52 integrated by homologous recombination into said target gene of said ES cells;

(d) introducing said transformed ES cells into a blastocyst;

(e) implanting said blastocyst into a pseudopregnant mouse; and

(f) allowing said embryo to develop to term, wherein only steps (a)-(c) are accomplished only once.

55. A recombinant nucleic acid molecule of claim 1, which is pMTI-2455, pMTI-2454, pMTI-2453, or pMTI-2398.