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Definitions

• the present invention relates to gene-targeted, non-human mammals comprising a human mutation in the non-human mammalian presenilin 1 (PS-1) FAD gene, methods of identifying compounds for treating Alzheimer's disease, and to methods of treating Alzheimer's disease.
• PS-1 non-human mammalian presenilin 1
• AD Alzheimer's disease
• Hallmark pathologies include the atrophy of brain gray matter as a result of the massive loss of neurons and synapses, and protein deposition in the form of both intraneuronal neurofibrillary tangles and extracellular amyloid plaques within the brain parenchyma.
• affected areas of the AD brain exhibit a reactive gliosis that appears to be a response to brain injury.
• Surviving neurons from vulnerable populations in AD show signs of metabolic compromise as indicated by alterations in the cytoskeleton (Wang et al. , Nature Med, 1996, 2, 871-875), Golgi complex (Salehi et al., J. Neuropath.
• AD Alzheimer's disease
• STM2 STM2 or "presenilin 2" (PS-2), located on chromosome 1
• PS-2 has been linked to two additional FAD kindreds including the descendants of German families from the Volga valley of Russia (Levy-Lahad et al. , Science, 1995, 269, 973-977; Rogaev et al. , Nature, 1995, 376, 775-778).
• PS-1 and PS-2 share an overall 67% amino acid sequence homology, and primary structure analysis indicates they are integral membrane proteins with 6 to 8 trans-membrane domains (Slunt et al. , Amyloid - Int. JExp. Clin. Invest.
• SEL-12 a 6 to 8 trans-membrane protein that appears to participate in an intracellular signaling pathway mediated by the lin-12/glp-l /Notch family (Levitan and Greenwald, Nature, 1995, 377, 351- 354).
• PS-1 and SEL-12 proteins share a 49% sequence homology and have similar membrane orientations.
• both human PS-1 and PS-2 can rescue the mutant sel-12 phenotype in C. elegans, indicating a role for the presenilins in Notch signaling (Levitan et al, Proc. Natl. Acad. Sci. USA, 1996, 93, 14940-14944).
• FAD linked to the presenilins is highly penetrant and the aggressive nature of the disease suggests that the mutant protein participates in a seminal pathway of AD pathology.
• PS-1 over seventy FAD mutations have been identified in PS-1, and three FAD mutations have been found in PS-2.
• Most of the FAD mutations occur in conserved positions between the two presenilin proteins, suggesting that they are affecting functionally or structurally important amino acid residues.
• many of the mutated amino acids are also conserved in SEL-12. All but two of the presenilin mutations are missense mutations.
• the presenilin proteins are processed proteolytically through two intracellular pathways. Under normal conditions, accumulation of 30 kD N-terminal and 20 kD C-terminal proteolytic fragments occurs in the absence of the full-length protein. This processing pathway is highly regulated and appears to be relatively slow, accounting for turnover of only a minor fraction of the full-length protein. The remaining fraction appears to be rapidly degraded in a second pathway by the proteasome (Thinakaran et al, Neuron, 1996, 17, 181-190; Kim et al, J. Biol. Chem., 1997, 272, 11006-11010). Proteolytic metabolism of PS- 1 variants linked to FAD appears to be different, but the relevance of the change to pathogenesis is not known (Lee, et al, Nature Med, 1997, 3, 756-760).
• mutant presenilins influence other AD pathogenic processes as well, such as presumptive intracellular signaling and cell death pathways involved directly or indirectly in neuronal dysfunction and degeneration.
• mice Genetically-engineered animals have been used extensively to examine the function of specific gene products in vivo and their role in the development of disease phenotypes.
• the genetic engineering of mice can be accomplished according to at least two distinct approaches: (1) a transgenic approach where an exogenous gene is randomly inserted into the host genome, and (2) a gene-targeting approach where a specific endogenous DNA sequence or gene is partially or completely removed, or replaced by homologous recombination.
• the transgene of a transgenic organism is comprised generally of a DNA sequence encoding the protein sequence and a promoter that directs expression of the protein coding sequences.
• a transgenic organism expresses the transgene in addition to all normally-expressed native genes.
• the targeted gene of a gene-targeted animal can be modified in one of two ways: (1) a functional form where a modified version of the targeted gene is expressed, or (2) a nonfunctional or "null" form where the targeted gene has been disrupted resulting in loss or reduction of expression. If the targeted gene is a single copy gene and the animal is homozygous at the targeted locus, then, depending on the type of modification, the animal either does not express the targeted gene or expresses only a modified version of the targeted gene in absence of the normal form.
• mice expressing native and mutant forms of the presenilin proteins have been described (Borchelt et al, Neuron, 1996, 17, 1005-1013; Duff et al, Nature, 1996, 383, 710-713; Borchelt et al, Neuron, 1997, 19, 939-945; Citron et al, Nature Med, 1997, 3, 67-72; Chui et al. , Nature Med. , 1999, 5, 560-564; and Nakano et al. , Eur. J. Neuroscl , 1999, 11, 2577- 2581). Although mice bearing mutations in PS-1 had elevated levels of A ⁇ l-42, they have not formed A ⁇ deposits characteristic of AD or shown behavioral deficits associated with AD.
• mice in which both PS-1 alleles have been disrupted resulting in the complete loss of PS-1 expression are not viable and die shortly after birth.
• No abnormal phenotypes or changes in APP processing have been reported in mice lacking only one of the two PS-1 alleles, but inhibition of APP processing is found in neurons derived from PS-1 null mice (DeStrooper et al, Nature, 1998, 391, 387-390).
• a human PS-1 mutation the P264L mutation in particular, was introduced into the mouse PS-1 gene.
• the P264L mutation is a non-conservative amino acid substitution in the cluster of mutations in exon 8, causing an onset of FAD in the middle forties to middle fifties (Campion et al, Hum. Molec. Genet., 1995, 4, 2373-2377; Wasco et al, Nature Med, 1995, 1, 848).
• Crosses produced APP ⁇ 1 ⁇ 1 ' x PS-l P264 ⁇ yp264L double gene-targeted mice. These mice had elevated levels of A ⁇ l-42, sufficient to cause A ⁇ deposition.
• mice bearing the PS-1 P264L mutation were also crossed with Tg2576 mice that overexpress Swedish APP695 (Hsiao et al, Science, 1996, 274, 99-102; available from the Mayo Clinic, Rochester, MN).
• One distinct advantage of the present invention is that for heterozygous and homozygous gene-targeted mice, the fidelity of expression patterns of proteins is maintained since the expression is under the endogenous promoter. Further, expression levels of the holoprotein are not changed.
• the present invention relates to a gene-targeted, non-human mammal comprising a gene encoding a mutant protein product of a mutated FAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation, and a humanized A ⁇ mutation, and generational offspring thereof.
• PS-1 mutated FAD presenilin-1
• the present invention also relates to a gene-targeted, non-human mammal comprising a gene encoding a mutant protein product of a mutated FAD presenilin-1 (PS-1) gene and a human Swedish APP695 mutation, and generational offspring thereof.
• the PS-1 gene has been mutated to contain the human P264L mutation (Wasco et al, Nature Medicine, 1995, 1, 848).
• the present invention relates to a mouse wherein a part of a mouse presenilin 1 gene encoding presenilin 1 protein has been replaced with a DNA sequence that results in a mouse presenilin 1 gene that contains a human mutation, most preferably a P264L mutation. Still more specifically, the base sequence of codon 264 of the mouse presenilin 1 gene is altered from CCG to CTT, which is the base sequence found to constitute the P264L mutation of humans. The mutated gene codon encodes leucine in place of proline in amino acid number 264 of presenilin 1.
• a nucleotide base in codon 265 of the mouse presenilin 1 gene is altered from adenosine to guanosine, but this change does not result in an amino acid change in the expressed protein.
• the present invention features a non-human mammal and generational offspring homozygous for a targeted mutant PS-1 gene comprising a mutated FAD gene preferably a mouse presenilin 1 protein-encoding sequence comprising a human mutation, most preferably a P264L mutation, in place of the native presenilin 1 protein-encoding sequence.
• a mutated FAD gene preferably a mouse presenilin 1 protein-encoding sequence comprising a human mutation, most preferably a P264L mutation, in place of the native presenilin 1 protein-encoding sequence.
• the present invention is also directed to methods for identifying a compound for treating Alzheimer's disease comprising administering a compound to a mammal heterozygous or homozygous for a mutation of the PS-1 gene and a human Swedish APP695 mutation, or generational offspring thereof, or to a mammal heterozygous or homozygous for a mutation of the PS-1 gene, a human FAD Swedish mutation, and a humanized A ⁇ mutation, and generational offspring thereof, and measuring the amount of A ⁇ 42 peptide in a tissue sample from the mammal.
• the present invention is also directed to methods of treating an individual suspected of having Alzheimer's disease comprising administering to the individual an effective Alzheimer's disease treatment amount of a compound identified by the method described above.
• the present invention is also directed to compounds identified by any of the methods described above.
• Figure 1 is a schematic diagram illustrating general principles of gene targeting.
• Figure 2 is a set of mouse PS-1 genomic clone maps prepared using the FlashTM Non- radioactive Gene Mapping Kit.
• Letter abbreviations for restriction endonucleases are as follows: E, EcoRl; X, Xbal; H, Hindlll; B, BamHI; Xh, Xhol.
• Figure 3 is a representative restriction map used to illustrate a FlashTM restriction mapping method.
• Figure 4 is a diagram illustrating the strategy for placing exons 7 and 8 on the restriction map of PS-1.
• Figure 5 is a pair of genetic maps illustrating the relationship between Exon 8 of PS-1 and the pPSl-8-TV replacement vector.
• Letter abbreviations for restriction endonucleases are as follows: E, EcoRl; X, Xbal; H, Hindlll; B, BamHI; Xh, Xhol, N, Notl.
• Figure 6 is a schematic diagram illustrating the construction of plasmid pPNTIox 2 .
• Figure 7 is a schematic diagram illustrating the construction of plasmid pPS 1 -XH16.
• Figure 8 is a schematic diagram illustrating the construction of plasmid pPSl-XBl.
• Figure 9 is a schematic diagram illustrating the construction of plasmids pPSl-X15 and pPSl-X2.
• Figure 10 is a schematic diagram illustrating the construction of plasmid pPSl-X319.
• Figure 11 is a schematic diagram illustrating the restriction mapping of the 5' Arm of
• Figure 12 is a pair of restriction maps for the PSl 3' and 5' arms of homology.
• Figure 13 is a partial sequence of exon 8 of PS-1 illustrating the base changes to effect the P264L mutation and the addition of the Aflll restriction endonuclease site of this invention.
• Figure 14 is a schematic diagram illustrating the construction of plasmid pPSl-XB85.
• Figure 15 is a schematic diagram illustrating the construction of plasmid pPS 1-206.
• Figure 16 is a schematic diagram illustrating the construction of plasmid pPSl-360.
• Figure 17 is a schematic diagram illustrating the construction of plasmid pPNT3'413.
• Figure 18 is a schematic diagram illustrating the construction of plasmid pPSl-8-TV.
• Figure 19 is a schematic diagram illustrating the strategy to detect homologous recombination within mouse PSl.
• Letter abbreviations for restriction endonucleases are as follows: E, EcoRl; X, Xbal; N Notl; H, Hindlll; B, BamHI; A, Apal; Af, Aflll; Sc, Seal; K,
• the present invention relates to a gene-targeted, non-human mammal (and generational offspring of such mammal) that contains in the non-human mammal's endogenous (i.e., native) genome presenilin 1 gene that comprises a human mutation, most preferably a human P264L mutation.
• the non-human mammal can also comprise a human FAD Swedish mutation and a humanized A ⁇ mutation.
• a non-human mammal can also comprise, in addition to the human PS-1 mutation, a human Swedish APP695 mutation.
• the gene-targeted, non- human mammal produces a mutated presenilin 1 protein instead of the presenilin 1 protein normally produced by the non-human mammal.
• Gene-targeted, non-human mammals homozygous for a presenilin 1 gene containing a human mutation, such as the human P264L mutation, produce the mutated presenilin 1 protein exclusively.
• Gene-targeted, non-human mammals heterozygous for a presenilin 1 gene containing a human mutation, such as the human P264L mutation produce both the mutated presenilin 1 protein and the native presenilin 1 protein.
• the gene-targeted, non-human mammal of this invention is a rodent, and more specifically a mouse.
• the non-human mammal of this invention is generated by gene targeting, as opposed to transgenic techniques, the mammal produces the mutated presenilin 1 protein exclusively by normal endogenous presenilin 1 protein expression mechanisms.
• the presenilin 1 protein is expressed from genes having the normal copy number, and under the control of the endogenous presenilin 1 gene expression control mechanisms.
• the presenilin 1 protein in the non-human animal of this invention is produced with the same developmental timing, same tissue specificity, and same rates of synthesis normally associated with native presenilin 1 protein in the wild-type, non-human mammal.
• the gene-targeted, non-human mammals of this invention may be used as tools or models to elucidate the role of PS-1 comprising a human mutation, preferably the human P264L mutation, in the pathology and symptomatology of AD. They may be used to elucidate the manner in which the human mutation, preferably the P264L mutation, increases the production of the amyloid protein A ⁇ 42.
• the term "increase" when used in the foregoing context means that the levels of A ⁇ 42 produced by the non-human mammals disclosed herein are elevated relative to wild-type controls.
• the non-human mammals of this invention and generational offspring also may be used as assay systems to screen for in vivo inhibitors and for discovering and testing the efficacy and suitability of putative chemical compounds for their ability to inhibit the formation, presence and deposition of excessive amounts of A ⁇ peptide in the brain tissues, other tissues and body fluids (e.g., blood, plasma, and cerebrospinal fluid), said method comprising the steps of: (a) administering said chemical compounds to a non-human mammal homozygous or heterozygous for a targeted mutant PS-1 gene comprising a human mutation, preferably the human P264L mutation, comprising: a mouse PS-1 peptide encoding sequence containing a human mutation, preferably the P264L mutation, in place of the native PS-1 peptide encoding sequence and (b) measuring the amounts of A ⁇ peptide in brain tissues, other tissues and body fluids (or some combination thereof) of said non-human mammal, at an appropriate interval of time after the administration of said chemical compounds.
• a ⁇ peptide means either A ⁇ 40 or A ⁇ 42 or fragments thereof.
• arms of homology means nucleotide DNA sequences in a targeting vector: (1) which have sufficient length and homology to provide for site-specific integration of part of the targeting vector into the target gene by homologous recombination; (2) in which, or between which are located one or more mutations to be introduced into a target gene; and (3) which flank a positive selectable marker.
• homologous recombination means rearrangement of DNA segments, at a sequence-specific site (or sites), within or between DNA molecules, through base-pairing mechanisms.
• human mutation in the non-human mammalian presenilin 1 (PS-1) FAD gene means any mutation of the PS-1 gene in a non-human mammal that results in the non-human mammal having a nucleotide or nucleotides that correspond to the human PS-1 gene at the corresponding position of the nucleotide or nucleotides.
• G209R which is disclosed in Sugiyama et al, Online Human Mutat, 1999, 14, 90, which is incorporated herein by reference in its entirety
• L219P which is disclosed in Smith et al, Neuroreport, 1999, 10, 503-507, which is incorporated herein by reference in its entirety
• M233L and A409T both of which are disclosed in Aldudo et al, Human Mutat, 1999, 14, 433- 439, which is incorporated herein by reference in its entirety
• E273A which is disclosed in Kamimura et al, J. Neurol.
• L282R which is disclosed in Aldudo et al, Neuroscl Lett., 1998, 240, 174-176, which is incorporated herein by reference in its entirety
• G378A which is disclosed in Besancon et al, Human Mutat, 1998, 11, 481, which is incorporated herein by reference in its entirety
• N405S which is disclosed in Yasuda et al. , J. Neurol. Neurosurg. Psychiatr.
• human P264L mutation means the following: the nucleotide sequence of codon 264 of the presenilin 1 gene is changed from CCG to a sequence selected from the group consisting of: CTT; CTC; CTA; CTG; TTA; TTG; and most preferably changed from CCG to CTT. Additionally, the nucleotide sequence of codon 265 of the presenilin 1 gene optionally, but preferably, is changed from AAA to AAG. The above described most preferable change of base sequences in codon 264 constitute the human P264L mutation. The optional, but preferred, change of the base sequence of codon 265 adds an Aflll cleavage site to the gene.
• target gene or “targeted gene” means a gene in a cell, which gene is to be modified by homologous recombination with a targeting vector.
• gene-targeted, non-human mammal means a non-human mammal comprising one or more targeted genes via a gene-targeting, as opposed to transgenic, approach.
• generational offspring in relationship to “gene-targeted, non-human mammal” means an animal whose genome includes the same gene-targeted manipulation as the parent(s) of that offspring.
• generational offspring For example, and not limitation, where a mammal whose genome has been manipulated by gene-targeting techniques to include a human mutation is then used for breeding purposes, all subsequent generations derived from that first mammal(s) are considered "generational offspring" so long as the genome(s) of such subsequent generational offspring comprises the gene-targeted manipulation as the original mammal; by design, this definition does not exclude other genomic-manipulations which may also be present in such generational offspring, nor does this definition require that such generational offspring be derived solely by cross-breeding techniques between a male and female mammal.
• transgenic non-human mammal means a non-human mammal in which a foreign ("transgene") gene sequence has been inserted randomly in a non-human mammal's genome and is therefore expressed in addition to all normally expressed native genes (unless the inserted transgene has interrupted a gene thus preventing its expression).
• targeting vector or "replacement vector” means a DNA molecule that includes arms of homology, the nucleotide sequence (located within or between the arms of homology) to be incorporated into the target gene, and one or more selectable markers.
• wild-type control animal means a non-gene-targeted, non-human mammal of the same species as, and otherwise comparable to (e.g., similar age), a gene-targeted non-human mammal as disclosed herein.
• a wild-type control animal can be used as the basis for comparison, in assessing results associated with a particular genotype.
• the first step in producing a gene-targeted non-human mammal of this invention is to prepare a DNA targeting vector.
• the targeting vector is designed to replace, via homologous recombination, part of the endogenous presenilin 1 gene sequence of a non-human mammal, so as to introduce the human mutation, preferably the P264L human mutation.
• the targeting vector is used to transfect a non-human mammalian cell, e.g., a pluripotent, murine embryo-derived stem (“ES") cell. In this cell, homologous recombination (i.e., the gene-targeting event) takes place between the targeting vector and the target gene.
• ES pluripotent, murine embryo-derived stem
• the mutant cell is then used to produce intact non-human mammals (e.g., by aggregation of murine ES cells to mouse embryos) to generate germ-line chimeras.
• the germline chimeras are used to produce siblings heterozygous for the mutated targeted gene.
• interbreeding of heterozygous siblings yields non-human mammals (e.g., mice) homozygous for the mutated target gene.
• Targeting vectors for the practice of this invention can be constructed using materials, information and processes known in the art. A general description of the targeting vector used in this invention follows.
• a targeting vector or replacement vector for use in this invention has two essential functions: (1) to integrate specifically (and stably) at the endogenous presenilin 1 target gene; and (2) to replace a portion of an exon of the endogenous presenilin 1 gene, thereby introducing the human mutation, and the mutation that introduces a new cleavage site in the gene.
• a portion of exon 8 is replaced so as to introduce the P264L mutation.
• the first basic structural feature of the targeting vector is a pair of regions, known as
• arms of homology which are homologous to selected regions of the endogenous presenilin 1 gene or regions flanking the presenilin 1 gene. This homology causes at least part of the targeting vector to integrate into the chromosome, replacing part (or all) of the presenilin 1 target gene, by homologous recombination.
• Homologous recombination in general, is the rearrangement of DNA segments, at a sequence-specific site (or sites), within or between DNA molecules, through base-pairing mechanisms.
• the present invention relates to a particular form of homologous recombination sometimes known as "gene targeting.”
• the second basic structural feature of the targeting vector consists of the actual base changes (mutation(s)) to be introduced into the target gene.
• the base changes in codon 264 of exon 8 resulted in an amino acid change in amino acid 264 from proline to leucine when the mutated gene was expressed to make protein.
• Other base changes can be made, as desired, to introduce any of the human mutations listed above into the mammalian genome.
• the mutation(s) to be introduced into the presenilin 1 target gene is located within the "arms of homology.”
• Gene targeting which affects the structure of a specific gene already in a cell, is to be distinguished from other forms of stable transformation, wherein integration of exogenous DNA for expression in a transformed cell is not site-specific, and thus does not predictably affect the structure of any particular gene already in the transformed cell.
• a reciprocal exchange of genomic DNA takes place (between the "arms of homology" and the target gene), and chromosomal insertion of the entire vector is advantageously avoided.
• the examples of this patent disclosure set forth the construction of a presenilin 1 gene targeting vector (and its use) to mutate the murine presenilin 1 protein encoding sequence so that it encodes the murine presenilin 1 protein, containing the human P264L mutation, or any of the other human mutations recited above, and an additional cleavage site.
• a presenilin 1 gene targeting vector and its use to mutate the murine presenilin 1 protein encoding sequence so that it encodes the murine presenilin 1 protein, containing the human P264L mutation, or any of the other human mutations recited above, and an additional cleavage site.
• targeting vectors can be designed to introduce the same mutations, using the principles of homologous recombination. Gene-targeted, non-human mammals produced using such other targeting vectors are within the scope of the present invention. A discussion of targeting vector considerations follows. The length of the arms of homology that flank the replacement sequence can vary considerably without significant effect on the practice of the
• the arms of homology must be of sufficient length for effective heteroduplex formation between one strand of the targeting vector and one strand of a transfected cell's chromosome, at the presenilin 1 target gene locus. Increasing the length of the arms of homology promotes heteroduplex formation and thus targeting efficiency. However, it will be appreciated that the incremental targeting efficiency accruing per additional homologous base- pair eventually diminishes and is offset by practical difficulties in target vector construction, as arms of homology exceed several thousand base pairs. A preferred length for each arm of homology is 50 to 10,000 base pairs.
• the arms of homology may lie within the presenilin 1 target gene, but it is not necessary that they do so and they may flank the presenilin 1 target gene.
• the targeting vector will comprise, between the arms of homology, a positive selection marker.
• the positive selection marker should be placed within an intron of the target gene, so that it will be spliced out of lnRNA and avoid the expression of a target/marker fusion protein.
• the targeting vector will comprise two selection markers; a positive selection marker, located between the arms of homology, and a negative selection marker, located outside the arms of homology.
• the negative selection marker is a means of identifying and eliminating clones in which the targeting vector has been integrated into the genome by random insertion instead of by homologous recombination.
• Exemplary positive selection markers are neomycin phosphotransferase and hygromycin ⁇ phosphotransferase genes.
• Exemplary negative selection markers are Herpes simplex thymidine kinase and diphtheria toxin genes.
• the positive selection marker can be flanked by short loxP recombination sites isolated from bacteriophage PI DNA. Recombination between the two loxP sites at the targeted gene locus can be induced by introduction of ere recombinase to the cells. This results in the elimination of the positive selection marker, leaving only one of the two short loxP sites. (See, U.S. Patent No. 4,959,317, which is herein incorporated by reference in its entirety). Excision of the positive selectable marker from intron 8 of the mutated presenilin 1 gene can thus be effected.
• Figure 1 illustrates the general principles of gene-targeting for introducing mutations into a mammalian genome using homologous recombination (reviewed in Capecchi, M. R, Trends Genet, 1989, 5, 70-76; Koller and Smithies, Ann. Rec. Immunol, 1992, 10, 705-730).
• a length of genomic DNA is first depicted by organizing it into regions (numbered 0-5 in Figure la).
• regions numbered 0-5 in Figure la
• several base pair changes (from 1-10) are to be incorporated into the cellular DNA around region 3.
• Homologous recombination using a gene targeting vector is utilized.
• the type of gene targeting vector used to incorporate these changes is termed a replacement vector.
• a "replacement vector” herein refers to a vector that includes one or more selectable marker sequences and two contiguous sequences of ES cell genomic DNA that flank a selectable marker. These flanking sequences are termed “arms of homology.” In Figure lb, the arms of homology are represented by regions 1-2 and 3-4.
• the use of DNA derived from the ES cells (isogenic DNA) helps assure high efficiency recombination with the target sequences (te Riele et al, Proc. Natl. Acad. Sci. USA, 1992, 89, 5128-5132).
• the arms of homology are placed in the vector on either side of a DNA sequence encoding resistance to a drug toxic to the ES cells (positive selection marker).
• a gene encoding susceptibility to an otherwise nontoxic drug is placed outside the region of homology.
• the positive selection marker is neo r , a gene that encodes resistance to the neomycin analog G418, and the negative selection marker is the herpes simplex virus thymidine kinase gene (HSN-tk) that encodes susceptibility to gancyclovir.
• HSN-tk herpes simplex virus thymidine kinase gene
• the positive selection marker is inserted into the genome between regions 2 and 3 in this example (making the transformed cells resistant to G418) while the negative selection markers is excluded (making the cells resistant to gancyclovir).
• transfected ES cells are grown in culture medium containing G418 to select for the presence of the neo r gene and gancyclovir to select for the absence of the HSN-TK gene.
• the positive selection marker is eliminated by using, for example, cre/lox technology once the mammal is crossed with another mammal.
• base pair changes are introduced into one of the arms of homology it is possible for these changes to be incorporated into the cellular gene as a result of homologous recombination.
• the mutations are incorporated into cellular D ⁇ A as a result of homologous recombination depends on where the crossover event takes place in the arm of homology bearing the changes. For example, as depicted by scenario "A” in Figure 1, the crossover in the arm occurs proximal to the mutations and so they are not incorporated into cellular DNA. In scenario “B”, the crossover takes place distal to the position of the mutations and they are incorporated into cellular DNA. Because the location of the crossover event is random, the frequency of homologous recombination events that include the mutations is increased if they are placed closer to the positive selection marker.
• the skilled artisan can achieve the incorporation of the selectable marker at a preselected location in the gene of interest flanked by specific base pair changes.
• the artisan would preferably choose to have the selectable marker incorporated within the intron of the gene of interest so as not to interfere with endogenous gene expression while the mutations would be included in adjacent coding sequence so as to make desired changes in the protein product of interest (Figure 1), (Askew et al, Mol. Cell. Biol, 1993, 13, 4115-4124, Fiering et al, Proc. Natl Acad. Sci. USA, 1993, 90, 8469-8473; Rubinstein et al, Nuc.
• a gene-targeted, non-human mammal comprising a human PS-1 mutation is prepared.
• the mammal can be heterozygous (contains one copy of the human PS-1 mutation) or homozygous (contains two copies of the human PS-1 mutation).
• a mouse is prepared which is PS-1 P264L/+ (heterozygous) or PS- lP264L/P 2 6 4 (hOHlOZygOUS).
• the gene-targeted, non-human mammals comprising a human PS-1 mutation described above can be crossed with mammals having a Swedish FAD mutation and "humanized" A ⁇ sequence in the APP gene (e.g., APP 10 ' 11 ⁇ 11 mouse) to produce mammals referred to as APP 1 ⁇ 1 ⁇ 11
• the present invention is also directed to a method for identifying a compound for treating Alzheimer's disease.
• a compound is administered to a mammal that is heterozygous or homozygous for a mutation of the PS-1 gene and also contains a human Swedish APP695 mutation, or generational offspring thereof, or to a mammal heterozygous or homozygous for a mutation of the PS-1 gene, a human FAD Swedish mutation, and a humanized A ⁇ mutation, and generational offspring thereof.
• Any compound to be tested can be administered in a variety of amounts by any variety of routes including, but not limited to, intravenously, orally, direct injection in the brain, and the like.
• a tissue sample from the mammal including, but not limited to, brain tissue, non-brain tissue and body fluids (e.g. blood and plasma) is obtained and the amount of A ⁇ peptide in the tissue sample is measured.
• a decrease in the amount of A ⁇ peptide in the tissue sample is indicative of a compound that can be used to treat Alzheimer's disease.
• the present invention is also directed to a method of treating an individual suspected of having Alzheimer's disease.
• An individual suspected of having Alzheimer's disease is any human having been examined by a physician and diagnosed as having Alzheimer's disease or symptoms thereof.
• a compound identified by the methods described above relating to a mammal that is heterozygous or homozygous for a mutation of the PS-1 gene and also contains a human Swedish APP695 mutation, or generational offspring thereof, or to a mammal that is heterozygous or homozygous for a mutation of the PS-1 gene, a human FAD Swedish mutation, and a humanized A ⁇ mutation, and generational offspring thereof, is administered to the individual in an amount effective to decrease the amount of A ⁇ peptide in the brain of the individual.
• An amount effective to decrease the amount of A ⁇ peptide can be determined from the identification process of the compound using a mammal that is heterozygous or homozygous for a mutation of the PS-1 gene and also contains a human Swedish APP695 mutation, or generational offspring thereof, or using a mammal that is heterozygous or homozygous for a mutation of the PS-1 gene, a human FAD Swedish mutation, and a humanized A ⁇ mutation, or generational offspring thereof, as a starting amount and scaling up for use in humans as is well known to those skilled in the art.
• An effective Alzheimer's disease treatment amount is an amount of a compound that measurably reduces the physiological pathology of Alzlieimer's disease or an amount that reduces the physical manifestations or symptoms of Alzheimer's disease.
• One skilled in the art can, for example, begin with an amount of a compound that decreases the amount of A ⁇ peptide in the brain, as described above, and can scale up or down the amount depending on the desired effect and the effect achieved in a particular individual.
• the present invention is also directed to compounds that are identified by the screening methods described above.
• the compounds can be any identifiable chemical or molecule, including, but not limited to, a small molecule, a peptide, a protein, a sugar, a nucleotide, or a nucleic acid, and such compound can be natural or synthetic.
• a small molecule including, but not limited to, a small molecule, a peptide, a protein, a sugar, a nucleotide, or a nucleic acid, and such compound can be natural or synthetic.
• the mouse PS-1 genomic DNA was cloned from a bacteriophage library created from 129/Sv mouse DNA partially digested with Sau3 A and into the BamHI site of Lambda DASHTM II (Reaume et al, Science, 1995, 267, 1831-1833, which is incorporated herein by reference in its entirety).
• a bacteriophage library created from 129/Sv mouse DNA partially digested with Sau3 A and into the BamHI site of Lambda DASHTM II (Reaume et al, Science, 1995, 267, 1831-1833, which is incorporated herein by reference in its entirety).
• PCR polymerase chain reaction
• DNA was prepared from bacteriophage particles purified on a CsCl gradient (Maniatis et al). Restriction maps were then generated for each of the cloned inserts using the FLASHTM Non-radioactive Gene Mapping Kit (Stratagene® Inc., La Jolla, CA). A typical restriction map generated by this method is illustrated in Figure 3. This method of restriction enzyme mapping involves first completely digesting 10 ⁇ g of the bacteriophage DNA with the restriction enzyme Notl using standard restriction enzyme digest conditions (Maniatis et al.) .
• Notl cuts all clones in the vector DNA at either end of the cloned insert so as to leave a T3 bacteriophage promoter attached to one end of the insert and a T7 bacteriophage promoter attached to the other end.
• the Notl digested DNA is then partially digested with the enzyme EcoRl, as an example, using limiting amounts of enzyme (0.2 units/ ⁇ g DNA) in an 84 ⁇ l reaction volume at 37°C. Aliquots (26 ⁇ l) were removed after 3 minutes, 12 minutes and 40 minutes and the digest reaction was stopped by the addition of 1 ⁇ l of 0.5 M EDTA.
• DNA from all three time points was resolved on a 0.7% agarose gel, visualized by ethidium bromide staining, and then transferred to a GeneScreen Plus® membrane (NEN® Research Products, Boston, MA) by capillary transfer (Maniatis et al, supra).
• the membrane was hybridized with an alkaline phosphatase labeled oligonucleotide that was specific for the T3 promoter (supplied with the FLASHTM kit) using reagents and methods supplied by the kit manufacturer. After hybridization, the membrane was washed and developed with a chemiluminescent-yielding substrate and then exposed to X-ray film in the dark for approximately 60 minutes.
• the oligonucleotide probes effectively label one end of the insert.
• Exon 8 was located on the restriction map hybridizing exon-specific probes to complete digests of each of the 6 different lambda genomic clones. Initially, 3 ⁇ g of DNA from each of the 6 different clones was completely digested with the restriction enzymes EcoRl and Xbal. The digested DNA was resolved on a 0.8% agarose gel, visualized by means of ethidium bromide staining and transferred to a GeneScreen Plus® membrane by capillary transfer. The membrane was then hybridized with a DNA probe that specifically hybridized to sequences from mouse PS- 1 exon 8.
• This probe was generated by PCR using oligonucleotides FEX8 (ATT TAG TGG CTG TTT TGT G; SEQ ID NO:3) and REX8 (AGG AGT AAA TGA GAG CTG GA; SEQ ID NO:4) which hybridize to the 5' and 3' ends of exon 8, respectively.
• FEX8 ATT TAG TGG CTG TTT TGT G
• REX8 AGG AGT AAA TGA GAG CTG GA; SEQ ID NO:4
• exon 7-specific probe was generated using PCR primers F892 (TGA AAT CAC AGC CAA GAT GAG; SEQ ID NO:5) and PS1-1 (GCA CTC CTG ATC TGG AAT TTT G; SEQ ID NO:6).
• Exon 7 was localized to the 2 kb Xbal-EcoRl fragment of all clones except ⁇ PSl-22 which allowed for the determination that exon 7 is located between positions 7.0 and 9.0 on the summary map ( Figure 2).
• Exon-specific probes were also used to obtain additional restriction map information using additional restriction enzymes. For example, when ⁇ PSl-22 was digested with Notl and BamHI, resolved on an agarose gel, transferred to a Genescreen Plus® membrane and probed with the exon 8-specific probe, a 700 bp fragment was identified. This information, when combined with the information from the other bacteriophage clones, allowed placement of the BamHI at position 11.7 on the composite map ( Figure 2). This process was repeated for the restriction enzyme Xhol.
• a 4.7 kb Xbal-BamHI fragment (which also contains two internal Xbal fragments) located at positions 7.0 to 11.7 on the summary map ( Figure 2), was chosen as the 5' arm that included the necessary mutations and a 4.1 kb BamHI-EcoRI fragment (which also contains an internal EcoRl site) located at positions 11.7 to 15.8 on the summary map ( Figure 2), as a 3' arm.
• the oligonucleotides PNT Not (GGA AAG AAT GCG GCC GCT GTC GAC GTT AAC ATG CAT ATA ACT TCG TAT; SEQ ID NO:7) and PNT Xlio (GCT CTC GAG ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAT GC; SEQ ID NO: 8) ( 50 ng of each) were combined in a 30 ⁇ l reaction mixture containing 5 U of Klenow polymerase, Klenow polymerase buffer and 2 mM dNTPs (dATP, dCTP, dGTP, and dTTP).
• plasmid DNA was isolated from ampicillin-resistant bacteria (Holmes et al,Anal Biochem., 1981, 114, 193-197) and analyzed by restriction enzyme analysis. The proper recombinant plasmids were identified as having acquired Sail, Hpal and Nsil sites (present in the linker) while still retaining the Notl and Xhol sites of the starting plasmid.
• pXN-4 Figure 6
• Plasmid DNA was digested with Xbal and BamHI, end-labeled with 32 P-dCTP and Klenow polymerase, and resolved on an 8% acrylamide gel (Maniatis et al). The gel was dried and exposed to X-ray film.
• Xbal-Hindlll fragment (positions 11.5 to 15.9 on the summary map, Figure 2) containing the 3' arm of homology and the fragment used for in vitro mutagenesis was first isolated from ⁇ PSl-22 by digesting 30 ⁇ l of the phage DNA with Xbal and Hindlll, resolving the digested DNA on a 0.8 agarose gel, visualizing the DNA with ethidium bromide staining and then excising the 4.4 kb fragment from the gel. DNA was purified from the gel using GeneClean® II (BiolOl Inc., La Jolla, CA).
• plasmid DNA was isolated from ampicillin-resistant bacteria and analyzed by restriction enzyme analysis to identify the resultant plasmids ( Figure 7).
• plasmid DNA from transformed bacteria was first analyzed by digesting it with Xbal and Hindlll in order to determine whether the plasmid DNA had acquired the 4.4 kb PS- 1 fragment. This plasmid was designated "pPS I-XH16.” Similar procedures were used to isolate a 200 bp Xbal-BamHI fragment from pPSl—
• each of the two plasmids were digested with the enzyme Hindi, resolved on an agarose gel, and visualized with ethidium bromide. Because a Hindi site is known to exist in the pBlueScrip® SK+ plasmid backbone within the multiple cloning site region near the T7 promoter relative to the insert position, it was possible to determine the position of the Hindi site in the 4.2 PS-1 fragment by determining the fragment sizes in each of the two digested samples ( Figure 11).
• a total of 3 base pair changes were introduced into the exon 8 region using a PCR strategy (for summary of changes, see Figure 13).
• the P264L mutation, and an Aflll site were introduced.
• Ten ng of pPSl-XBl were included into each of two PCR reactions.
• the first reaction contained the primers EXPL2 (TTG TGT CTT AAG GGT CCG CTT CGT ATG; SEQ ID NO:l 1) and T7 (Stratagene Cloning Systems, La Jolla, CA). This generated a 220 bp band that encompassed the 3' end of exon 8 and clone PS1-XB1.
• This fragment also included the P264L mutation and a novel Aflll site that resulted as part of the P264L change.
• the second PCR reaction used the primers EXPL1 (CGG ACC CTT AAG ACA CAA
• AAC AGC CAC SEQ ID NO: 12
• T3 Program Cloning Systems, La Jolla, CA. This generated a 137 bp fragment that encompassed the 5' end of exon 8 and PSl -XB1. This fragment also included the P264L change and an Aflll site ( Figure 14).
• the product of the first reaction was purified using MagicTM PCR Preps DNA Purification System (Promega Corporation, Madison, WI) and digested with BamHI and Aflll in order to liberate the restriction sites at its ends.
• the product of the second reaction was purified and digested with Aflll and Xbal. These two fragments, as well as Xbal and BamHI digested pBlueScript® SK+ were ligated together and transformed into HB101 competent E. coli cells. The DNA was isolated and analyzed from the ampicillin resistant colonies.
• pPSl-XB85 The clone bearing a recombinant plasmid in which the two PCR fragments had joined together at their Aflll site and inserted into the BamHI and Xbal sites of pBlueScript® SK+ was called pPSl-XB85 ( Figure 14).
• direct nucleotide sequencing was performed using T3 and T7 sequencing primers (Stratagene Inc., LaJolla, CA) and Sequenase Version 2.0 DNA Sequencing Kit (United States Biochemical, Cleveland, OH).
• the 5' arm of homology was assembled in pBlueScript® SK+ through several cloning steps. First, pPSl-XB15 was partially digested with Xbal so that only one Xbal site was cut. The resulting DNA was then digested with BamHI and gel purified ( Figure 15).
• the mutated insert in pPSl-XB85 was released by digesting it with Xbal and BamHI and gel purifying the resulting mutated insert.
• the 200 bp Xbal-BamHI fragment was ligated into the linearized pPSl-X15 and recombinant plasmids were screened for the proper orientation of the insert by means of an Aflll digest.
• the correctly oriented plasmid yielded 1.9 kb and 5.8 kb fragments. This plasmid was designated "pPS 1-206."
• pPS 1 -206 was linearized by a partial Xbal digest ( Figure 16).
• the Xbal fragment from pPSl-X319 was isolated and cloned into the linearized pPS 1-206 DNA.
• Orientation of the 300 bp Xbal fragment was determined by sequencing the recombinant clone as well as ⁇ PSl-20 with primer EX8PL1 using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Life Science Inc., Cleveland, OH).
• the plasmid pPNTIox 2 was prepared for receiving the 3' arm of homology by first digesting plasmid DNA with EcoRI and BamHI and gel isolating the linear plasmid ( Figure 17). In parallel, the 3' arm was prepared by partially digesting pPSl-XH16 with EcoRI and isolating the linear form. This fragment was then digested with BamHI and the 4.1 bp fragment gel isolated. The 3' arm was ligated to pPNTIox 2 . The resulting plasmid was designated "pPNT3'413.” The 5' arm was inserted into pPNT3'413 to give the final plasmid pPSI-8-TV.
• the 5' arm was liberated from plasmid DNA by first digesting with Xhol and Notl.
• pPNT3'413 was prepared by double digesting with Notl and Sail The two fragments of DNA were ligated and transformed into competent WM 1100 E. coli cells ( Figure 18).
• Example 3 Mutagenesis of the Mouse PS-1 Gene in ES cells
• the Rl line of ES cells derived from 129/Sv x 129/Sv-CP FI hybrid mice was utilized. These cells were grown in ES cell medium consisting of Dulbecco's Modification of Eagle's Medium (with L-glutamine and 4500 mg/L glucose; Mediatech Inc., Herndon, VA) supplemented with 20% fetal bovine serum (FBS; Hy clone Laboratories Inc., Logan, Utah; cat.
• the cultures were passed every 48 hours or when the cells became about 80% confluent.
• the cells were first washed with phosphate buffered saline (without Ca 2+ and Mg 2+ ) and then treated with a trypsin/EDTA solution (0.05% trypsin, 0.02% EDTA in PBS without Ca 2+ and Mg 2+ ). After all of the cells were in suspension, the trypsin digestion was stopped by the addition of tissue culture medium. The cells were collected by centrifugation, resuspended in 5 ml of tissue culture medium and a 1 ml aliquot of the cell suspension was used to start a new plate of the same size.
• pPSl-8-TV DNA 400 ⁇ g was prepared for electroporation by digesting it with Notl in a 1 ml reaction volume. The DNA was then precipitated by the addition of ethanol, washed with 10% ethanol and resuspended in 500 ⁇ l of sterile water. The Notl-linearized pPS 1 -8-TV DNA was electroporated into ES cells using a Bio-Rad
• the cells in a 24-well plate were first chilled by placing the plate on ice. The medium was then replaced with fresh ES cell medium supplemented with 10% DMSO and 25% FBS and the plate was cooled at approximately 0.5°C/min by insulating the plate in a styrofoam box and placing it in a -70°C freezer.
• the medium in each well was replaced with 500 ⁇ l of digestion buffer (100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 ⁇ g/ml proteinase K). After overnight incubation at 37°C, 500 ⁇ l of isopropanol was added to each well and the plate was agitated for 15 minutes on an orbital shaker. The supernatant was aspirated and replaced with 500 ⁇ l of 70% ethanol and the plate was shaken for an additional 15 minutes.
• digestion buffer 100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 ⁇ g/ml proteinase K.
• the DNA precipitate was picked out of the well and dissolved in 50 ⁇ l of TE solution (10 mM Tris-HCl pH 7.5, 1 mM EDTA).
• the primary analysis for mutagenesis of the mouse PS-1 gene involved a Southern hybridization screen of Apal digested ES cell DNA.
• the probe for this analysis was derived from the 3' end of our cloned PS-1 region outside of the 3' arm of homology ( Figure 19d). It was prepared by first isolating the 6 kb Xbal fragment corresponding to the 3' end of ⁇ PSl-6 ( Figure 2) and subcloning it into Xbal digested pBlueScript® SK+. A further digest of this subclone, called pPSl-X6 with Xhol (an internal site) and Hindlll (from the Bluescript® S K+ polylinker) yielded the 1000 bp probe.
• the 15 kb band represents the unaltered cellular copy of PS-1 while the 9 kb band is derived from the PS-1 copy in which the novel Apal site results in a shorter fragment.
• 8 cell lines were identified as potential targeted cell lines out of 260 cell lines analyzed.
• PSI + normal PS-1 gene
• PS1-87 The mutagenized form of the PS-1 gene described here has been called, PS1-87, PS1-175, PS1-176, and PS1-243. Three of these lines were thawed, cell numbers expanded, and used to establish PS-1 mutant mice.
• Additional mutagenesis of the mouse PS-1 gene can be performed in ES cells in the manner described above in order to comprise other human mutations.
• PS-1 mutant ES cells were used to make chimeric mice by aggregating the mutant ES cells to E2.5 embryos and transferring the aggregated embryos to pseudopregnant females (Wood et al, Nature, 1993, 365, 87-89). ES cells were prepared for aggregation by limited trypsinization to produce clumps that average 10-15 cells. E2.5 embryos were collected from superovulated CD-I female mice by oviduct flushing as described by Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, 1986, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
• the zona pelucida was removed from the embryos using acidic Tyrode's solution (Sigma Chemical Co., St. Louis, MO). Aggregation wells were created by pressing a blunt metal instrument (a darning needle) into tissue culture plastic. Embryos were then placed in a well together with a clump of approximately 10-15 ES cells in a small drop (approximately 20 ⁇ l) of M16 medium (Sigma Chemical Co., St. Louis, MO) under mineral oil. After an overnight incubation (37°C, 100%) humidity, 5% C0 2 in air) the aggregate embryos were transferred to the uterine horns of a pseudopregnant female (Hogan et al, 1986, supra).
• ES cells Contribution of the ES cells to the offspring was scored by the appearance of pigmented coat color. Positive mice are termed chimeric founders. Germline contribution by the ES cells was scored by the appearance of pigmented offspring from a cross between the chimeric founders and CD-I females.
• PCR primers were as follows: neo28 (GGA TTG CAC GCA GGT TCT CC; SEQ ID NO: 13); and neo445 (CCG GCT TCC ATC CGA GTA CG; SEQ ID NO: 14).
• the genomic DNA was prepared from a tail sample (Hogan, 1986, supra).
• one female mouse was heterozygous for ps-l 1 ⁇ 2641 - (PS-l nP264L + ), i.e., this mouse was positive for the neo r cassette based upon the foregoing PCR strategy.
• Subsequent generational offspring which are also heterozygous for pS-l nP264L have been developed by mating of this female with wild-type males.
• mice heterozygous for PS-1" P264L were genotyped using a PCR-based method.
• the presence of the wild-type allele for murine PS-1 was scored using the following primers: X8F (CCC GTG GAG GAG GTC AGA AGT CAG; SEQ ID NO: 15) and X8R (TTA CGG GTT GAG CCA TGA ATG; SEQ ID NO:16). Scoring with these primers yields a 142 bp fragment (data not shown).
• the presence of the mutant allele was scored using the primers neo28 and neo445, which yields a 417 bp fragment.
• mice which are heterozygous for the mutation yield both bands; mice which are homozygous for the mutation yield only the 417 bp band; and mice that are homozygous for the wild-type allele yield only the 142 bp band.
• Tissue samples were derived from animal tails, and the PCR procedures of Example 1 were utilized for such scoring.
• mice homozygous for the PS-l nP264L allele were generated by cross breeding of heterozygous mice (PS-l nP264L + ) with mice which are homozygous for a humanized APP gene (as disclosed in PCT Publication Number W096/34097, published October 31, 1996; incorporated herein fully by reference).
• the resulting generational offspring were then determined to be heterozygous for both the ps-l 1 * 2641 - allele and heterozygous for the humanized APP gene (data not shown); these generational offspring were then utilized for cross-breeding, with resulting generational offspring determined (using the PCR procedure outlined above) to be homozygous for the PS-l nP264L allele, as well heterozygous for the humanized APP gene (generational offspring from this liter were also found to be heterozygous for the ps-l nP264 allele/homozygous for the humanized APP gene; and heterozygous for the ps-l nP264L allele/heterozygous for the humanized APP gene ⁇ due to the limited number of pups obtained from this liter, double homozygotes were not found). Subsequent matings produced PS- 1 «P 2 6 4 L «P264L ⁇ A ppN h/N h mice
• mice homozygous for the PS-P P264L allele were also generated by cross-breeding of heterozygous mice (PS-l nP 64L/+ ). In one set of matings, 6 homozygotes were found amongst 27 offspring, which is well within the expected 25% recovery of homozygotes from a heterozygous cross. Accordingly, and based upon the various breeding approaches disclosed above, substantially normal viability and embryonic survival of the animals is evident.
• Example 6 Excision of the FGK-neo cassette pBS185 plasmid DNA (Sauer et al, New Biol, 1990, 2, 441-449, incorporated herein by reference in its entirety) encoding Cre recombinase was introduced by pronuclear injection into one-cell embryos generated from a ps-l nP264L + x CD-I cross. Since the plasmid was circular, DNA integration into the genome had a very low frequency of occurrence. Transient expression of the DNA to produce Cre recombinase excised the PGK-r ⁇ eo cassette in the early embryos. Injected embryos were transferred to pseudopregnant females. Excision of the PGK-r ⁇ eo cassette was confirmed by genotyping of the offspring. These mice were designated PS-1 P264L+ and were crossed to generate PS-1 P264L/P264L mice.
• the VGK-neo gene was excised by recombination at the flanking loxP sites after transient expression of Cre recombinase.
• the loss of the PGK-Reo gene was scored in the offspring as a 219-base pair fragment by PCR using the X8F and X8R primer pairs as described in Example 5.
• the mutant PS-1 allele with the neomycin- selectable marker excised was designated PS-1 P264L .
• PS-1 P264L/+ mice were crossed with Tg2576 mice and APP ⁇ 1 ⁇ 11 mice to further study the effects of the P264L mutation on A ⁇ production and deposition.
• PS-1 + + , PS-l nP264L nP264 , and ps-l P264L P264L mice, aged 2-6 months were used for evaluating mRNA and protein levels of PS- 1.
• Total RNA was extracted from one-half brain by homogenization in RNAzol B (Tel-Test, Friendswood, TX).
• Messenger RNA was selected with Oligotex columns (Qiagen, Valencia, CA).
• Equal volumes of mRNA were mixed with loading buffer (NormernMAX-Gly, Ambion, Austin, TX) heated to 50°C for 30 min, separated on a 0.7% agarose gel, and transferred to a nylon membrane.
• PS-1 mRNA was detected with a 32 P-dUTP- labeled riboprobe representing the 3' end of human PS-1 : nucleotides 1083-1428 cloned into a pGEM-T vector (Promega, Madison, WI). The same blot was hybridized with a GAPDH probe (Ambion) for normalization. To visualize mRNAs, the membrane was exposed to a phosphor screen, scanned on a Storm 840 Phosphorlmager, and densitometry performed with ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).
• One-half brain was homogenized in 2.5 ml of buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS, 0.25% deoxycholate, 0.25% NP-40, and protease inhibitors (5 mM PMSF, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml pepstatin) (Lee et al, Nature Med, 1997, 3, 756-760). Protein concentration was determined by BCA assay (Pierce, Rockford, IL). Total brain ly sates were mixed with reducing loading buffer and heated at 37°C for 45 min.
• B17.2 was raised against amino acid residues 300-315 (EGDPEAQRRVSKNSKY; SEQ ID NO:17) in the hydrophilic loop domain of human PS-1.
• the N-terminal fragment was detected with CP160 at 1 :500.
• CP160 was generated using a 6X-histidine tagged N-terminal fragment of human PS-1 (amino acids 1-80), expressed in bacteria with a pQE-9 plasmid (Qiagen).
• the synthetic PS-1 N-terminal fragment was purified using Ni-NTA agarose (Qiagen) and SDS-PAGE. Peptide was cut out of acrylamide gels for injection into rabbits.
• the IgG fraction of CP160 was affinity purified and used for blotting.
• the primary antibodies were detected with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (New England Biolabs, Beverly, MA). Blots were reacted with chemiluminescent reagent (LumiGLO, New England Biolabs) and exposed to Hyperfilm (Amersham, Arlington Heights, IL). Films were scanned and densitometry performed with RFLP2.1 software (Scanalytics, Fairfax, VA).
• PS-1 protein levels were reduced by about 50% (data not shown). Both the N- and C-terminal fragments were reduced to a similar degree. These data indicate that the PS-1 P264L mutation affects PS-1 protein levels either via effects on translation, processing of the full length PS-1 protein, or stability of the cleaved fragments.
• PS-1 FAD mutations were found to cause no reductions in fragment formation in FAD patients, in transfected cells, and in transgenic or gene-targeted mice (Hendriks et al, NeuroReport, 1997, 8, 1717-1721;' Lee, et al, Nature Med, 1997, 3, 756-760; Podlisny et al, Neurobiol Dis., 1997, 3, 325-337; Guo et al, Nature Med, 1999, 5, 101-106; Levesque et al, Molec.
• Example 8 A ⁇ 40- and 42-specific ELISAs
• Half brains from predepositing double gene-targeted mice (APP NLh/NLh mice at 1-6 months, APPTM 1 TM 1 x PS-1 P264 + mice at 5 months, and APPTM" 1 x PS-1 P264L P264 mice at 1-2 months) and predepositing Tg2576 mice (Hsiao et al, Science, 1996, 274, 99-102) at 2-4 months were frozen on dry ice and stored at -70°C.
• the supernatants were neutralized to pH 8 with 2 M Tris-HCl, assayed for protein concentration by the BCA method (Pierce, Rockford, IL), and diluted 1 :1 in 5% fetal clone serum (HyClone, Logan, UT) and 1% nonfat dry milk in TBS.
• the A ⁇ 42-specific EL ⁇ SA was run as previously described (Savage et al, J. Neuroscl, 1998, 18, 1743-1752). The A ⁇ 40-specific ELISA was modified (Savage et al, J.
• Table 2 shows the effect of the PS-1 P264 mutation on A ⁇ 40 and A ⁇ 42 levels in the brains of APP NLh/NLh mice before the appearance of A ⁇ deposition.
• the PS-1 P264 mutation did not have a significant effect on the level of A ⁇ 40.
• One copy of the PS-1 P264L mutation slightly elevated A ⁇ 42 but the effect of the mutation was significant only in the APP NLh/NLh x PS- I P264L P264 mice compared with the APP NLh/NLh x PS-1 + + mice.
• Table 3 shows the effect of the PS-1 P264L mutation on A ⁇ 40 and A ⁇ 42 levels in the brains of Tg2576 mice before the appearance of A ⁇ deposition.
• the PS-1 P264L mutation did not have a significant effect on the level of A ⁇ 40.
• One copy of the PS-1 P26 L mutation slightly elevated A ⁇ 42 but the effect of the mutation was significant only in the Tg2576 x ps-l P264L P264L mice compared with the Tg2576 x PS-1 +/+ mice.
• the increase in A ⁇ 42 levels caused a significant elevation in the ratio of A ⁇ 42 to A ⁇ 40 in the Tg2576 x ps-l P264 ⁇ yp264L mice relative to Tg2576 x PS-1 +/+ mice.
• the effect of the PS-1 P264 mutation on A ⁇ levels was similar for the Tg2576 and APP h/NLh mice.
• Example 9 Immunohistochemistry and Histology ps _ ⁇ P264L/P264 mice were examined at 12 months of age. APP ⁇ 11 mice that were PS- 1 +/+ , PS-1 P264L + , or ps-l P264L p264L , aged 3, 6, 9, 12, 15, and 18 months of age were evaluated. Additional mice examined were Tg2576 and were PS-1 +/+ , PS-1 P264L + , or PS-1 P264L/P264L , aged 1, 2, 4, 6, 9, 12, 15, and 18 months of age. Other Tg2576 mice maintained by crossing to C57B6/SJL mice were also examined at 6, 9, 12, 15, 18, and 21 months of age.
• mice were perfused with Ringer' s solution and the brains removed and hemisected. One-half of each brain was immersed in 70% ethanol and 150 mM NaCl for 48 hours, paraffin embedded and sectioned in the sagittal plane at 10 ⁇ m. Sets of 16 sections taken at intervals of 200 ⁇ m were stained to demonstrate A ⁇ deposits by immunohistochemistry.
• Antibodies used were 1153, a rabbit polyclonal antibody generated against amino acids 1-28 of human A ⁇ (Savage et al, Neuroscience, 1994, 60, 607-619) and monoclonal antibodies 4G8 and 6E10 (Senetek).
• Sections were pretreated with 80% formic acid for 4G8, not pretreated for 1153 and 6E10, and were reacted with the primary antibodies overnight at 1:1,000. Antibodies were complexed using biotinylated secondary antibodies (1 :100), linked using streptavidin labeled horseradish peroxidase (BioGenex, San Ramon, CA), and visualized using nickel-intensified 3,3'- diaminobenzidene. Non-transgenic mice, as well as pre-absorbed primary antisera, served as staining controls. Additional sets of sections were stained using thioflavine S and examined with a fluorescence microscope.
• Plaque load was quantified in neocortex in one set of 16 sections stained with antibody 1153 using the CastGrid system (Olympus, Copenhagen, Denmark). Volume of neocortex and percent volume of neocortex occupied by A ⁇ deposits were determined stereologically by point counting (Weibel et al, (1979) Stereological methods, vol. 1 : practical methods for biological morphometry, 415 pp. London: Academic Press). Representative results are shown in Table 4.
• Tg2576 x PS-l P264 ⁇ y+ mice A ⁇ deposition was not noted at 2 months of age but was present at 4 months of age.
• Tg2576 x PS- ⁇ P264L P264L mice A ⁇ deposition was not present at 1 month of age but was present at 2 months of age.
• Tg2576 X PS-1 + + mice did not show A ⁇ deposition until 6 months of age, and the amount was comparable to that seen in 6-month-old Tg2576 mice maintained on the C57B6/SJL background.
• a ⁇ plaque load visualized with antibody 1153 increased dramatically in neocortex of the Tg2576 x PS-1 P26 L + and Tg2576 x ps-l P264L P2 ⁇ 4L mice at later ages (data not shown).
• a ⁇ deposition in the APP NLh/N h mice has been assessed out to 22 months of age. No evidence for deposition was found. Similarly, no deposition was found in ps-l p264 P264L mice that were wild type for mouse APP at 12 months of age. Extremely rare A ⁇ deposition was noted in the cortex of APP 1 TMTM 1 x PS-1 P264L + mice using both antibody 1153 and thioflavine S at 12 months of age. At 18 months of age A ⁇ deposits in these mice were more numerous and larger.

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