JP2001525168A - vector - Google Patents

Abstract

  (57) [Summary] YAC vectors are described that can be used individually or in combination to prepare YACs. The first YAC vector comprises a nucleotide sequence comprising a sequence represented as SEQ ID NO: 1 or SEQ ID NO: 4 or a variant, homolog or derivative thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to vectors, especially vectors suitable for use as or with yeast artificial chromosomes or in the preparation of yeast artificial chromosomes.

[0002] Yeast artifical chromosome (also known as YAC) contains structural components of the yeast chromosome into which very large amounts of DNA can be cloned. As a specific example, it is generally possible to clone into the DNA of a YAC portion of about 1000 kb in length, which can be a plasmid (typically a DNA portion of up to 20 kb), a bacteriophage λ (typically Can be cloned in other cloning vectors such as cosmids (typically DNA fragments of up to 45 kb) and cosmids (typically DNA fragments of up to 100 kb). It is much larger than the DNA part (Lodish 1995 Molecular Cell Biology 3rd Edition, Pub.
ientific American Books, Pge 233).

[0003] The YAC was first published by Burke et al (Burke, DT, GFCarle and MVOlson (1987) Clon
ing of large segments of exogenous DNA into yeast by means of artificial
chromosome vectors. Science 236: 806-812). A general introduction to YAC can be found in TA Brown 1995 (Gene Cloning, An Introduction, 3rd Edition,
page 325, Pub. Chapman & Hall, pages 139-142).

[0004] Typically, a YAC contains the following essential functional components: centromere, two telomeres, and one or more origins of replication. Centromeres are needed to properly position chromosomes in daughter cells during cell division, telomeres are needed to ensure proper replication, and origins of replication ensure that DNA replication begins. To exist. The origin of replication is often called the ARS (autonomous replication sequence) component.

[0005] YACs are typically made from YAC vectors. These vectors are typically circular. If these vectors are needed for use in YAC production, they are linearized, for example, by using specific restriction enzymes.

[0006] To date, many YAC vectors have been proposed in the literature. Examples of such vectors include pYAC2 and pYC discussed in U.S. Pat. No. 4,889,806.
AC4. Other YAC vectors are disclosed in WO-A-95-03400. Other YAC vectors include pYAC3 and pYAC5.

[0007] YAC vectors such as pYAC3, pYAC4, pYAC5 are essentially pBHR322 plasmids into which many yeast genes have been inserted. These genes are located in the yeast centromere region (CE
It contains two telomeres (called TEL) and two selectable marker genes (called URA3 and TRP1). The TEL sequence does not correspond to the complete genomic telomere sequence. Nevertheless, these subsequences still function as telomere sequences. One origin of replication (called ori, eg, ARSs1) is located between CEN4 and TRP1. When used for cloning, the YAC vector is cut with BamHI and another restriction enzyme (SmaI for pYAC3, EcoRI for pYAC4, NotI for pYAC5). These fragments are then ligated with the nucleotide sequence of interest (called "NOI" for ease of calling) digested with the corresponding restriction enzymes.

The resulting linear structure is shown in FIG. 17 (the figure is not drawn to scale). The resulting linear structure -YAC- contains the essential functional components of the chromosome in the correct orientation.

[0009] YAC has been used for mapping the human genome. It is still used today. In this case, the NOI is a gene or a fragment thereof that has not been completely sequenced (or functioned). In these studies, YACs are used to create genomic libraries that are then screened. As an example, the physical map of the human Y chromosome and the long arm of chromosome 21 has been determined by analysis of long fragments of human DNA cloned into YAC, especially by the site where the sequence was labeled. This study is summarized in Lodish et al 1995 (ibid., P. 285).

However, the use of YAC is not limited to mapping the human genome. For example, a map of the Drosophila X chromosome containing the shibire (shi) locus was created by the use of YAC (Bliek and Meyerowitz 1991 Nature 351 441). YACs have also been proposed for mapping plant genomes, such as the Arabidopsis genome.

In addition to its use in genome mapping, YAC currently has another important application. In this regard, it has recently been found that under certain conditions, YACs can be introduced into mammalian cells (such as murine cells), where they behave functionally similar (or very similar) to endogenous chromosomes. Was. In this regard, it is possible to introduce YACs containing large fragments of human DNA (ie NOI) into mouse germ cells using cell fusion, microinjection or transfection techniques. This was initially achieved using a YAC containing a 670 kb human X chromosome fragment (Jacobovitis et al 1993 Nature 362 pages 255-258). As a further example, WO-A-94 / 23049 reports a YAC containing a gene encoding a β-amyloid precursor protein.

[0012] Thus, researchers are now able to analyze the in vivo functional behavior of NOIs, such as human NOIs, with YACs. Prior knowledge of the sequence and / or function of the NOI is not required for these studies.

An overview of these in vivo functional studies is described in Jacovovitis (Current Biology 1994 vol 4 N
o 8 pages 761-763), which states, `` The ability to replace mouse genes with their human equivalents using yeast artificial chromosome technology has provided a powerful tool for studying the regulation and function of human genes. New technology will be offered. "

[0014] Other reviews and details on these studies and techniques can be found in Larin and Lehrach (Gene
t. Res.Camb 1990 56 pp 203-208), Schedl et al (Nucleic Acids Research vo
l 20 No. 20 pages 3073-3077) and Montoliu et al (Reprod. Fert. Dev. 1994)
7 577-584).

[0015] YACs can also be used to study the functional behavior of mutant genes. In this regard, WO-A-95 / 14769 reports a method for producing mice that express human mutein sequences. This uses the “pop-in / pop-out” method in combination with YAC technology to insert mutations into YAC and thereby obtain stem cells that can be used for transgenic mouse development. are doing. This particular method involves obtaining the gene contained in the YAC, introducing a predetermined mutant human DNA sequence (NOI) into the YAC by homologous recombination, and suddenly utilizing the transgenic form. Inserting the mutant gene into embryonic stem cells and injecting the stem cells into scutellum cells to obtain transgenic mice expressing the mutant protein sequence. “Pop-in / pop-o
ut ”method is described in Rothstein (1991 Methods In Enzymology vol 194, Guide To Yeast G
enetics and Molecular Biology, Eds. Gutherie et al, San Diego: Academi
c Press. Pages 281-301) and McCormic et al (TCM Vol6 No. 1 1996 pages 16-
24).

According to Schedl et al (cited reference 28), the first report on yeast artificial chromosomes was immediately followed by the interest in introducing YAC DNA into mammalian cells. In successful transgenic cells, the genes contained in the YACs are embedded in almost native chromosomes, ensuring regulation of expression comparable to their endogenous counterparts. Thus, such a method should allow for more rapid identification of genes through complementation analysis and detailed studies on gene function and their regulation in vivo. Schedel et al. (Ibid.) Report a method for isolation of purified and concentrated YAC DNA and a protocol for microinjection into somatic cells and fertilized mouse oocytes in culture.

Although YACs have many important applications, there are problems associated with their production and use.

For example, when compared to conventional vector DNA (10% YAC), the lower efficiency of transgenic animals produced using YAC DNA is likely due to the lower concentration of YAC DNA available for injection. Due to that. 2 pl of 500 kb Y at a concentration of 1 ng / μl
Assuming that AC is injected into the pronucleus, only one molecule of YAC DNA is introduced into the fertilized egg. By amplification of yeast artificial chromosomes in yeast, YAC transgenic animals (i.e.
High concentration of YAC DNA increases production success rate of transgenic mammals including YAC)
Should be provided. However, to date no comprehensively acceptable solution to this problem has been found.

For example, Smith et al proposed the use of the YAC vector pCGS990 (Smith et al
Mammalian Genome 1993 4 pages 141-147). Even though the YAC vector is a way to overcome this problem, it contains the TK gene (ie, the herpes simplex virus thymidine kinase gene) as a selectable marker. This is problematic because expression of this gene can cause male infertility in transgenic animals.

[0020] Alternatively or additionally, it is not currently possible to simply monitor the in vivo expression pattern of NOIs introduced into organisms such as mice by current technology. Current techniques such as in situ hybridization, polymerase chain reaction (PCR), and Northern blot analysis are difficult to perform.

Further, as a further example, earlier reported studies have reported co-expression of NOI and reporter genes in YACs.

As described above, known vectors for producing YAC have problems. The present invention improves upon existing technologies associated with making and utilizing YACs. In this regard, the present invention provides two vectors that can be used alone or in combination with each other, thereby overcoming at least one of the above problems.

In accordance with a first aspect of the invention, there is provided one or more of the following examples, which are presented in numbered paragraphs for clarity.

1. YAC vector containing IRES. 2. A YAC vector containing a reporter gene capable of generating a visually detectable signal from the expression product of the reporter gene. 3. A YAC vector containing a reporter gene whose expression product can generate an immunologically detectable signal. 4. A YAC vector containing an IRES and a reporter gene, wherein the expression product of the reporter gene can generate a visually detectable signal.5. A YAC vector containing an IRES and a reporter gene, A YAC vector whose expression product of a reporter gene can generate an immunologically detectable signal. 6. pYIV1. 7. pYIV2. 8. pYIV3. 9. pYIV4. 10. YAC produced by (eg, by insertion of) any one of the vectors of the above examples. 11. Use of IACs to modify YAC vectors or YACs.

In accordance with a second aspect of the present invention, there is provided one or more of the following examples, which are presented in numbered paragraphs.

1. A YAC vector comprising a nucleotide sequence comprising the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4, or a variant, homolog or derivative thereof. 2. A vector capable of modifying a YAC or YAC vector, comprising a nucleotide sequence comprising the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4, or a variant, homolog or derivative thereof. 3. pYAM4. 4. YAC produced by the vector of any one of the above examples. 5. Use of a nucleotide sequence comprising the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4, or a variant, homolog or derivative thereof, in the vector to make a YAC vector or YAC. 6. In order to increase the expression efficiency of one or more NOIs in the YAC vector or YAC, the vector contains the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4, or a mutant, homolog or derivative thereof. Use of nucleotide sequences.

For convenience, the vectors of the first aspect of the invention are often referred to herein as insertion vectors, and the vectors of the second aspect of the invention are often referred to herein as amplification vectors.

However, the term “vector” as used herein in a general sense means a vector according to the first aspect of the invention and / or a vector according to the second aspect of the invention.

According to a third aspect of the invention, there is provided a combination of at least one embodiment of the first aspect of the invention with at least one embodiment of the second aspect of the invention. .

With respect to the third aspect of the invention, ie the combination of the vector of the first aspect of the invention and the vector of the second aspect of the invention, the term “combination” refers to the resulting Y
It means that an AC or transgenic / transformed cell, organ or organism is made by using both the vector of the first aspect of the invention and the vector of the second aspect of the invention.

The third aspect of the present invention relates to the production of a transgenic / transformed cell, organ or organism, as well as a YAC, the vector of the first aspect of the present invention and the vector of the second aspect of the present invention. It is not limited to production techniques where both of the vectors must be used simultaneously. In this regard, it is often necessary for the vectors of the first aspect of the invention that they be used at a different stage from the vectors of the second aspect of the invention when producing YACs or transgenic / transformed cells, organs or organisms. It is advantageous.

According to a further aspect of the present invention there is provided a YAC vector or YAC comprising a selection gene, which is specifically removable from the YAC vector or YAC.

Other aspects of the invention include the following. The NOI expression pattern can be determined by measuring a detectable signal (such as a visually or immunologically detectable signal) produced by the expression product of the reporter gene. YAC transgenic mammals that co-express genes.

A YAC transgenic mammal expressing a reporter gene under the control of a human NOI regulatory sequence. Use of YAC transgenic mammals to test the potential of pharmaceuticals or veterinary drugs.

Administering a substance to a YAC transgenic mammal of the invention, wherein the substance modifies a NOI or EP (“expression product”) (to affect expression pattern or activity) Determining whether the substance has a potential to influence the NOI expression pattern or its EP by a detectable signal.

[0036] To screen for a substance useful in treating any one of disorders of circadian function, sleep disorders, eating disorders, premenstrual syndrome, autoimmune diseases, birth defects in women, and / or sexual dysfunction. Of the present invention.

A substance identified by the method of the present invention.

(A) performing the analysis method of the present invention; (b) identifying one or more substances that affect the NOI expression pattern or its EP activity; Preparing the above identified substance.

(A) performing the analysis method of the present invention; (b) identifying one or more substances that influence the NOI expression pattern or its EP activity; and (c) one or more substances. Preparing a pharmaceutical composition selected from the group consisting of preparing the identified substances.

(A) performing the analysis method of the present invention; (b) identifying one or more substances that affect the NOI expression pattern or its EP activity; and (c) NOI expression. Modifying one or more identified substances that cause different effects on the pattern or its EP activity.

Use of any one of the YACs of the invention or the vectors of the invention for screening for substances that may affect the NOI expression pattern or its EP activity.

An in vitro assay according to the assay method of the present invention in the preparation of a pharmaceutical composition for treating a disease or condition associated with the NOI expression pattern or its EP activity.
Use of substances that affect the NOI expression pattern or its EP activity.

Use of a substance identified by the analytical method of the present invention in the manufacture of a medicament that affects the expression pattern of NOI or its EP activity.

Use of a substance identified by the analytical method of the present invention in the manufacture of a medicament that affects the expression pattern of NOI or its EP activity.

In accordance with the present invention, at least a portion of the analysis method can be performed on a living tissue.

A specific example of a preferred NOI is human serotonin transporter (SERT), preferably human serotonin transporter (SERT) represented as SEQ ID NO: 2 or a mutant, homolog or derivative thereof. It is not limited to these.

A “variant”, “homologue” or “homolog” in the context of the present invention.
The term "derivative" refers to a sequence that provides a resulting expression product of a nucleotide sequence having the same activity as the expression product of SEQ ID NO: 2, preferably at least as active as the expression product of SEQ ID NO: 2. One (or more) of the nucleic acids, including any of substitutions, changes, modifications, exchanges, deletions or additions. In particular, the term "homolog" covers identity with respect to structure and / or function if the resulting nucleotide sequence has promoter activity. Sequence identity (
(I.e. similarity), preferably have at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably it has at least 95%, more preferably at least 98% sequence identity. These terms also include allelic variations in the sequence.

[0048] Although not limited, examples of another preferred NOI is VIP ₂ receptor (VIPR2). The VIP2 receptor is referred to throughout the text as the VIP2 receptor, VIPR2 or VPC2R (Harmar et al 1998 P
harmacological Reviews 50: 265-270). Preferably, VIPR2 is SEQ ID NO: 3 or a variant thereof, VIP ₂ receptor expressed as homologues or derivatives (VIP
R2).

The terms “variant”, “homolog” or “derivative” in the context of the present invention have the same activity as the expression product of SEQ ID NO: 3, preferably at least at the same level as the expression product of SEQ ID NO: 3 Includes any substitution, alteration, modification, exchange, deletion or addition of one (or more) nucleic acids of the sequence that provides the resulting expression product of the active nucleotide sequence. In particular, the term "homolog" covers identity with respect to structure and / or function if the resulting nucleotide sequence has promoter activity. With respect to sequence identity (ie, similarity), it preferably has at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably it has at least 95%, more preferably at least 98% sequence identity. These terms also include allelic variations in the sequence.

The present invention relates to modified YACs comprising these points of the invention, transgenic cells comprising these points of the invention, transgenic organisms comprising these points of the invention, all of these points. Also included are the steps of preparing and methods of expressing all of these aspects.

The term “influence” is any of treating, inhibiting, suppressing, alleviating, restoring, regulating, affecting or altering an existing condition.
Including one or more.

The term “substance” includes any entity capable of affecting the expression pattern of the NOI or its EP activity (eg, including peptide sequences and variants / homologues / derivatives / fragments thereof). One or more compounds). Includes analogs, equivalents and mutants thereof. Also includes agonists, antagonists and antibodies. Non-limiting antibodies include polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by Fab expression libraries, and humanized monoclonal antibodies.

The term “expression product” or “EP” refers to the expressed protein itself, but also includes fusion proteins that include all or part of the same. An EP is the same as a naturally occurring form or a variant, homolog, or derivative thereof.

The term “disorder of circadian function” refers to physical patterns that can occur throughout extreme conditions in dementia, such as work patterns such as shift work, normal aging, time lag travel (jet fatigue). Disorders that can cause psychiatric disorders.

There are many advantages associated with the present invention. For example, the use of an amplification vector makes it possible to obtain many copies of YAC.

For example, by using an insertion vector, the in vivo expression pattern of NOI can be easily monitored.

In addition, by using an insertion vector, YAC is contained in YAC or as YAC,
The NOI and the reporter gene can be expressed so as to overexpress it.

The present invention provides a means for producing YAC transgenic animals, and a method for analyzing these animals in terms of the expression, regulation, and function of NOI present in YAC DNA.

According to the present invention, it is easy to determine the site / region in which NOI is expressed and to identify substances that may affect the NOI expression pattern or its EP activity.

Other advantages associated with the present invention will become apparent from the following text. In the context of the present invention, a vector contains at least one NOI. In the context of the present invention, the term NOI (ie, the nucleotide of interest) includes all suitable nucleotide sequences and need not necessarily be a natural DNA sequence. Thus, DNA sequences can be, for example, synthetic DNA sequences, recombinant DNA sequences (ie, produced using recombinant DNA technology), cDNA sequences or partial genomes, and include combinations thereof. The DNA sequence need not be a coding sequence. DNA
If the sequence is a coding sequence, it need not be the entire coding region. Further, the DNA sequence may be in the sense or antisense direction. Preferably, it is the sense direction. Preferably, the DNA is a cDNA.

The vectors of the present invention can be used to make modified YACs or modified YAC vectors. Modified YACs or modified YAC vectors are, for example, NO
It can be used to study expression and / or regulation and / or function of I. In addition, the vectors of the present invention can be used in combination with other materials, such as other NOIs, compounds or compositions, to modify or modify YACs or modifications that can be used to study NOI expression and / or regulation and / or function. It can be used to produce a modified YAC vector. In addition, modified YACs or modified YAC vectors can be used to test the potential of pharmaceuticals (including veterinary drugs).

[0062] As used herein, the term "modified YAC or modified YAC vector" refers to a modified YAC or modified YAC vector having a modified genetic structure. With respect to the present invention, after some or all of the vectors of the present invention have been incorporated into a YAC or YAC vector, the YAC or YAC vector has a modified genetic structure.

The vector of the present invention can be used, for example, to produce a transformed cell that can be used for studying the function of NOI. In addition, the vectors of the present invention can be used in combination with other materials, such as other NOIs, compounds or compositions, to produce transformed cells that can be used to study NOI function. In addition, transformed cells can be used to test the potential of pharmaceuticals (including veterinary drugs).

As used herein, the term “transgenic organism” refers to an organism that contains a modified genetic structure. With respect to the present invention, after the vector of the present invention has been introduced into an organism, the organism has a modified genetic structure.

The term “organism” includes all suitable organisms. In a preferred embodiment, the organism is a mammal. In a highly preferred embodiment, the organism is a mouse.

For some applications, transgenic animals can be produced by utilizing the transformed cells of the present invention.

In a preferred embodiment, the insertion vector of the invention is itself a YAC vector. For some applications, the amplification vector of the invention itself is a YAC vector.

In the context of the present invention, the vector of the present invention may further comprise one or more selection genes, whereby the vector and any product comprising or made from the same (for example, It allows selective growth and / or screening of modified YAC vectors, YACs or specific yeast strains containing any one of the same. These selection genes can be selected from the available selection genes.
Examples of suitable selection genes include LYS2 (Barnes and Thorner 1986, Mol and Cell B
iol 6: pp2828-2838), LEU2 (Beach and Nurse 1981 Nature vol 290 pp140-
142) and ADE2 (see Stotz and Linder 1990 Gene 95 pp91-98). In a preferred aspect, any one or more of the selection genes can be specifically removed from the vectors, modified YAC vectors and modified YACs of the invention. In this regard, the term "specifically removable" refers to the ability to remove one or more selected genes without disrupting other regions in the vectors, modified YAC vectors and modified YACs of the present invention. Means For example, a selection gene can be flanked by unique restriction sites. Alternatively, the selection gene can be flanked by LoxP components that can be removed by Cre recombinase. For information on the use of LoxP components and Cre recombinase, see Deursen et al (1995 PNAS Vol 93 pages 7376-7380), K.
uhn et al (1995 Science Vol 269 pages 1427-1429) and Araki et al (1995 PN
AS Vol 92 pages 160-164). As a specific example, the selection gene flanked by LoxP components can be removed before or after the formation of the transgenic animal stem cells. Removal of the selection gene is highly desirable. This is because the transgenic organism does not express the selection gene, and thus the selection gene cannot affect the organism, nor can it affect the expression of the NOI being studied. Furthermore, removing the selection gene means that the NOI is closer to the 3 'regulatory region present in YAC.

For the purposes of the present invention, a YAC vector can further include one or more NOIs. The NOI need not be known in function and / or structure. Preferred NOIs are of human origin. According to one aspect of the invention, the YAC vector contains an internal ribosome entry site (internal
ribosomal entry site, or IRES).

A review of IRES can be found in Mountainford and Smith (TIG May 1995 vol 11 No. 5 pages 179-18).
It is reported in 4). Appropriate IRES is also available from Mountford et al (Mountford et al 1994 PNA
S 91 pages 4303-4307).

The IRES sequence has also been reported in WO-A-93 / 03143, WO-A-97 / 14809, WO-A-94 / 24301, WO-A-95 / 32298 and WO-A-96 / 27676 . These references do not disclose or suggest the use of IRES for the preparation of YACs, or that they are part of YACs.

According to WO-A-97 / 14809, in contrast to the effect of a promoter on the transcription of DNA,
The IRES sequence improves certain transcription efficiencies. Many different IRES sequences are known,
Encephalomyocarditis virus (EMCV) (Ghattas, IR, et al., Mol.Cell.Biol., 11: 5848-5
859 (1991), BiP protein [Macejak and Sarnow, Nature 353: 91 (1991)]; Drosophila antennapedia genes (exons d and e) [Oh, et al., Genes
& Development, 6: 1643-1653 (1992)] and IR of poliovirus
ES sequence (Pelletier and Sonenberg, Nature 334: 320-325 (1988); Mounford and Sm
ith, TIG 11,179-184 (1985)].

According to WO-A-97 / 14809, IRES sequences are typically found in the 5 ′ non-coding region of a gene. In the same article, in addition to these, IRES sequences look for gene sequences that affect expression, and then that sequence is DNA (ie, acts as a promoter or enhancer) or RNA only (ie, acts as an IRES sequence). Can be found empirically by deciding whether to affect

Accordingly, the present invention is not intended to be limited to any particular IRES sequence. Rather, any sequence can be used that can act as an IRES sequence—that is, one that can improve the efficiency of RNA translation.

A preferred IRES sequence is SEQ ID NO: 1 or a variant, homolog, derivative or fragment thereof.

The terms “variant”, “homolog” or “derivative” in the context of the present invention refer to the same activity as the expression product of SEQ ID NO: 1, preferably at least at the same level as the expression product of SEQ ID NO: 1. Includes any substitution, alteration, modification, exchange, deletion or addition of one (or more) nucleic acids of the sequence that provides the resulting expression product of the active nucleotide sequence. In particular, the term "homolog" covers identity with respect to structure and / or function if the resulting nucleotide sequence has promoter activity. With respect to sequence identity (ie, similarity), it preferably has at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably it has at least 95%, more preferably at least 98% sequence identity. These terms also include allelic variations in the sequence.

Another preferred IRES sequence is SEQ ID NO: 4 or a variant, homologue, derivative or fragment thereof.

The terms “variant”, “homolog” or “derivative” in the context of the present invention have the same activity as the expression product of SEQ ID NO: 4, preferably at least at the same level as the expression product of SEQ ID NO: 4 Includes any substitution, alteration, modification, exchange, deletion or addition of one (or more) nucleic acids of the sequence that provides the resulting expression product of the active nucleotide sequence. In particular, the term "homolog" covers identity with respect to structure and / or function if the resulting nucleotide sequence has promoter activity. With respect to sequence identity (ie, similarity), it preferably has at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably it has at least 95%, more preferably at least 98% sequence identity. These terms also include allelic variations in the sequence.

[0079] For any of SEQ ID NOs: 1-4, sequence identity is such that a simple "exhaustive" comparison of one or more sequences to another (i.e., an exact comparison)
One can determine whether it has at least 75% sequence identity to one or more sequences.

Mutual sequence identity can also be determined by commercially available computer programs that can calculate percent identity (%) between two or more sequences using, for example, default parameters and an algorithm appropriate for identity determination. Can decide. A typical example of such a computer program is CLUSTAL. Advantageously, the parameters are set to default values and the BLAST algorithm is used. The BLAST algorithm is available at http: //www.ncbi.
It is described in detail in gov / BLAST / blast-help.htm, which is hereby incorporated by reference. The search parameters are defined as follows and can be set to the defined default parameters.

The BLAST (Basic Local Alignment Search Tool) is a program that uses the programs blastp, blastn,
blastx, tblastn, a heuristic search algorithm used by tblastx (see http: //www/ncbi.nih.gov/BLAST/blast-henp; html):
Significance is sought from their search results using the Karlin and Altschl statistical method with some extensions. The BLAST program was adjusted for sequence similarity searches, eg, to confirm homology with the query sequence. For a discussion of the fundamental issues in similarity searching of sequence databases, see Altschl et al (19
94) See Nature Genetics 6: 119-129.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the following tasks:

[0083] Blastp compares an amino acid query sequence against a protein sequence database.

Blastn compares nucleotide query sequences against a nucleotide sequence database.

[0085] blastx searches a protein sequence database for nucleotide query sequences.
Compare the conceptual translation products of the six reading frames (both strands).

[0086] tblastn also compares protein query sequences against a dynamically translated nucleotide sequence database in all six reading frames (both strands).

[0087] tblastx compares six open reading frame translations of a sequence, which may be referred to as nucleotides, to six reading frame translations of a nucleotide sequence database.

BLAST uses the following search parameters: HISTOGRAM displays a histogram of scores for each search. Default is yes. (See parameter H in the BLAST manual.)

DESCRIPTIONS limits the number of short descriptions of matched sequences reported to the specified number. The default limit is 100 descriptions. (See parameter V on the manual page).

EXPECT is the threshold for the statistical significance of the number of matches reported against the database sequence. The default value is 10. This was set up by the stochastic model of Karlin and Altschul (1990) to be expected to find 10 matches by mere chance. If the statistical significance of a match is greater than the EXPECT threshold, the match is not reported. The lower the EXPECT threshold, the more rigorous and the fewer chance matches reported. Fractional values are also acceptable. (See the parameters in the BLAST manual.)

CUTOFF is a cutoff score of a high scoring segment pair. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database only if their statistical significance is at least as high as a single HSP with a score equal to the CUTOFF value. The higher the CUTOFF value, the less rigorous the chance of reporting an accidental match is. (See parameters in the BLAST manual). Typically, significance thresholds can be more intuitively handled with EXPECT.

ALIGNMENTS restricts database sequences to a specified number of high scoring segment pairs (HSPs) reported. The default limit is 50. If more database sequences meet the reported statistical significance threshold (see EXPECT and CUTOFF below), only the match with the greatest statistical significance is reported. (See BLAST manual parameter B).

MATRIX specifies an alternative scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff
, 1992). Valid alternative choices include PAM40, PAM120, PAM250 and IDENTITY. There is no alternative scoring matrix for BLASTN. Return to error response by specifying MATERIX index in BLASTN request.

STRAND limits TBLASTN searches to above or below database sequences. Or BLA
Limits STN, BLASTX or TBLASTX searches to open reading frames on the strand above or below the query sequence.

FILTER is a segment of a low-complexity query sequence determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17: 149-163, or Claverie & States (1993) Computers and Chemistry 17: 191- For 201 XNU programs, or for BLASTN, Tatusov and Lipman's DUST program (http
(see http://www.nocbi.nlm.nih.gov) to block segments consisting of short-period inner repeats. Filtering can reduce blast output statistically significant but not biologically meaningful reports (e.g., eliminate common acidic, basic or proline-rich regions) and identify specific database sequences Can leave more biologically interesting regions of the query sequence available for matching.

[0096] Low complexity sequences found by the filter program are replaced with the letter "N" in nucleotide sequences (eg, "NNNNNNNNNNNNNN") and replaced with the letter "X" in protein sequences (eg, " XXXXXXXXX ”).

[0097] Filtering is applied only to the query sequence (or its translation product), not to the database sequence. The default filtering is DUST for BLASTN and SEG for other programs.

When applied to sequences in SWISS-PROT, it is not uncommon for nothing to be occluded by SEG, XNU, or both, so one should not consider filtering to always work. In addition, in some cases, the entire sequence may be completely occluded, which means that the statistical significance of the reported matches for unfiltered sequences is questionable.

The NCBI-gi allows the NCBI-gi identifier to be displayed in the output in addition to the accession and / or place name.

Most preferably, sequence comparisons are performed using the simple BLAST search algorithm provided by http: //www.ncbi.nlm.nih.gob/BLAST.

Other computer programming methods for determining the identity and similarity of two sequences include the GCG program package (Devereux et al 1984 Nucleic Acids Research 1
2: 387) and FASTA (Atschul et al 1990 j Molec Biol 403-410).
It is not limited to these.

In some aspects of the invention, gap penalties are not used in determining sequence identity.

The present invention also encompasses nucleotide sequences that are complementary to the sequences set forth herein, or fragments or derivatives thereof. If the sequence is complementary to the fragment, the sequence can be used as a probe to identify similar promoters in other organisms.

The present invention also encompasses nucleotide sequences capable of hybridizing to the sequences set forth herein, or fragments or derivatives thereof.

Hybridization refers to “the process by which one strand of a nucleic acid binds to a complementary strand by base pairing” (Coombs J (1994) Dictionary of Biotechnology, Stockton Press,
New York NY) and Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Labo
ratory Manual, Cold Spring Harbor Press, Plainview NY) means the amplification process performed in the polymerase chain reaction technique.

Nucleotide sequences that are capable of hybridizing to the nucleotide sequences described herein under moderate to maximum stringency are also within the scope of the invention.
Hybridization conditions were determined by Berger and Kimmel (1987, Guide to Molecular Cloni
ng Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego
Based on the melting point (Tm) of the nucleic acid binding complex as reported in (CA), "stringency" is defined as described below.

The maximum stringency typically occurs at about Tm-5 ° C. (5 ° C. below the Tm of the probe). High stringency is about 5 ° C to 10 ° C below Tm, and moderate stringency is about 1 ° C below Tm.
Occurs at 0 ° C to 20 ° C lower. As will be apparent to those skilled in the art, maximum stringency hybridization can be used to identify or detect identical nucleotide sequences, with moderate stringency identifying or detecting similar or related nucleotide sequences. Can be used for

In a preferred aspect, the invention includes nucleotide sequences that are capable of hybridizing with the nucleotide sequences of the invention under stringent conditions (eg, 65 ° C., 0.1 × SSC).

The invention also encompasses nucleotide sequences that are capable of hybridizing to sequences complementary to the sequences provided herein, or fragments or derivatives thereof. Similarly, the present invention includes nucleotide sequences that are complementary to sequences capable of hybridizing to the sequences of the present invention. These types of nucleotide sequences are examples of various nucleotide sequences. In this regard, the term "diversified" includes sequences that are complementary to sequences capable of hybridizing to the sequences presented herein. However, preferably, the word `` various '' refers to the stringent conditions of the nucleotides provided herein (e.g., 0.1 × SSC ｛1 × SSC = 0.15 M NaCl, 0.015 Na Na citrate pH 7. 0｝) contains a sequence complementary to the hybridizable sequence.

The insertion of an IRES into a YAC by utilizing the insertion vector of the present invention, and expressing the modified YAC by forming at least one modified YAC, the expression of at least two nucleotide sequences-especially overexpression. It is possible to do. One of the two nucleotide sequences may be a NOI, such as a human-derived NOI. One of these nucleotide sequences can be another NOI. Alternatively, in a preferred respect, the other nucleotide sequence is a reporter gene of the present invention.

The present invention also includes a vector comprising one or more IRESs or a YAC obtained therefrom. In this embodiment, the vector or YAC obtained therefrom is preferably N
Including non-OI.

In a preferred aspect of the first aspect of the present invention, the YAC vector comprises a reporter gene whose expression product is capable of generating a visually detectable signal.
The following are specific examples of such a reporter gene. LacZ (Mansour et
al 1990 PNAS vol 87 pp7688-7692), green fluorescent protein (Chiocchetti et
al 1997 Biochem Biophys Acta 1352: pp 193-202; and Chalfie et al 1994 Sc
ience vol 263 pp 802-805), chloramphenicol acetyltransferase (Gorman et al 1982 Mol Cell Biol 2 (9) pp1044-1051; and Frebourg
and Brison 1988 Gene vol 65 pp 315-318) or luciferase (de Wet et
al 1987 Mol Cell Biol 7 (2) pp725-737; and Rodriguez et al 1988 PNAS vol
85 pp 1667-1671).

[0113] In another preferred embodiment of the first aspect of the present invention, the YAC vector has an expression product capable of generating an immunologically detectable signal or an immunologically detectable signal. Includes a reporter gene that is detectable by a substance that can supply a signal.

In a preferred aspect, the reporter gene, when fused to the NOI, has a hemagglutinin tag (see Pati 1992 Gene 15; 114 (2): 285-288), a c-myc tag (Emrich et al.
1993 Biochem Biophys Res Commun 197 (1): 214-220) or FLAG epitope (For
d et al 1991 Protein Expr Purif Apr; 2 (2): 95-107) to produce a fusion protein that can be detected by a commercially available antibody.

[0115] By using the reporter gene of the present invention, the function of NOI contained in a YAC library such as a YAC human DNA library can be easily observed.

For example, if the NOI in YAC has a role in regulating expression (like a promoter),
The reporter gene of the present invention is expressed by the transgenic organism of the present invention,
Researchers can easily determine the site or region where the expression regulatory component has activity. In addition, researchers can easily test substances and the like that may affect the expression ability or pattern of the regulatory component.

According to a further embodiment, if the NOI in YAC has a functional role other than the role of regulating expression, by utilizing the insertion vector of the present invention, the researcher may be able to (one or more spacing nucleotides). The NOI can be fused to the reporter gene of the invention (directly or indirectly by means such as sequence). Thus, if the NOI is fused to the reporter gene of the present invention or is present in the transgenic organism of the present invention, the researcher can easily determine the site or region where the NOI is expressed. it can. In addition, researchers can easily test substances that could affect the NOI expression pattern. A further advantage is that α can be easily monitored for NOI expression patterns or levels, allowing researchers to determine phenotype.

One aspect of the present invention pertains to the use of SEQ ID NO: 1 or SEQ ID NO: 4 or a variant, homolog or derivative thereof. In this case, the term "mutant, homolog or derivative thereof" includes all additions, substitutions or deletions of one or more nucleic acids, provided that the resulting substance still functions as an IRES.

In a preferred aspect of the invention, all variants, homologues or derivatives of the IRES sequence comprise at least 100 bp SEQ ID No. 1 or SEQ ID No. 4. Preferably, the variants, homologues or derivatives all comprise at least 200 bp of SEQ ID NO: 1 or SEQ ID NO: 4. Preferably, the variants, homologues or derivatives all comprise at least 300 bp of SEQ ID NO: 1 or SEQ ID NO: 4. Preferably, all variants, homologues or derivatives comprise at least 400 bp SEQ ID NO: 1 or SEQ ID NO: 4. Preferably, the variants, homologues or derivatives all comprise at least 500 bp of SEQ ID NO: 1 or SEQ ID NO: 4. Preferably, there is at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity with the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4. Sex, preferably at least 95% sequence identity, preferably at least 98% sequence identity.

[0120] Preferably, the nucleotide sequence is the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4.

As indicated, the YAC vectors of the invention include SEQ ID NO: 1 or SEQ ID NO: 4, or a variant, homolog or derivative thereof. In this case, the nucleotide sequence enhances the expression efficiency of one or more NOIs in the YAC vector or YAC.

[0122] YAC vectors further include one or more marker genes. These genes can be selected from the available suitable marker genes. One example of a suitable marker gene is PG
K-Hyg (see Nara et al 1993 Curr Genet 23 (2): pp134-140).

The nucleotide sequence of the present invention can be used to modify a YAC or YAC vector such as pYAC1, pYAC2, pYAC3 or pYAC4. The present invention also includes a combination of the above aspects.

The following samples were obtained on November 24, 1997, in accordance with the Budapest Treaty:
National Collections of Industrial and Marine Bacteria Limited (NCIMB), 23 S
Deposited at t.Machar Drive, Aberdeen, Scotland, United Kingdom, AB21RY by the MRC Brain Metabolism Unit, Edinburgh Royal Hospital, Morningsaide Park, Edinburgh, EH105HF.

JM109pYIV1- deposit number NCIMB 40907 JM109pYIV2- deposit number NCIMB 40908 JM109pYIV3- deposit number NCIMB 40909 JM109pYIV4- deposit number NCIMB 40910 JM109pYAM4- deposit number NCIMB 40906

The present invention is described by way of example with reference to the following figures. FIG. 1 is a diagram of pYIV1. FIG. 2 is a diagram of pYIV2. FIG. 3 is a diagram of pYIV3. FIG. 4 is a diagram of pYIV4. FIG. 5 is a photograph. FIG. 6 is a diagram of pYAM4. FIG. 7 is a photograph of the gel. FIG. 8 is a photograph. FIG. 9 is a photograph. FIG. 10 shows the nucleotide sequence. FIG. 11 is a photograph of the gel. FIG. 12 is a PCR map integrating SERT 35D / D6 YAC DNA. FIG. 13 is a PCR map integrating VIPR2 HSC7E526 / V12 YAC DNA. FIG. 14 is a photograph. FIG. 15 is a photograph. FIG. 16 is a graph showing β-Gal activity determined using a chemiluminescence reporter assay system. FIG. 17 is a schematic diagram.

This will be described in some detail below. FIG. 7 shows amplification of YAC DNA by pYAM. S. cerevisiae endogenous chromosomal DNA is shown in lane 3, the size is shown in kb on the left. D in all other lanes
The NA plug-type Lys + YAC clone was loaded, and after retrofitting with pYAM4, was cultured in a medium containing galactose without lysine. YA in each clone
The movement position of the CDNA is indicated by an arrow. YAC DNA amplification levels are based on a comparison of ethidium bromide staining of endogenous chromosomes of similar size to YAC DNA.
Lane 1, 350kb, 8x, Lane 2, 630kb, 3x, Lane 4, 615kb, 3x, Lane 5,
500kb, 4x, Lane 6, 200kb, 5x, Lane 7, 200kb, 6x, Lane 8, 230kb, 2x, Lane 9, 150kb, 3x, Lane 10/11/12, 230kb, 5x.

FIG. 10 shows the IRES sequence obtained from encephalomyocarditis virus. The sequence has been genetically modified at the 3 'end to introduce a HindIII restriction site.

FIG. 14 shows immunohistochemical staining of the LacZ reporter gene in the suprachiasmatic nucleus of a transgenic mouse expressing YAC containing the human VPAC2R gene. It should be noted that single cell resolution is possible by immunohistochemical methods.

FIG. 15 shows histochemical staining for β-galactosidase activity in transgenic mice containing YAC HSC7E526 / V12.

FIGS. 15a and 15b show the staining patterns of β-galactosidase activity in coronal sections of the brain of transgenic mice paternal to mouse A108.2. In (a), the stained suprachiasmatic nucleus is indicated by the tip of the arrow, and in (b), an enlarged view is shown. FIG. 15c shows the same transgenic mouse (tg) and wild type (wt) littermate pancreatic staining.

FIG. 16 shows the tissue distribution of β-galactosidase activity in transgenic mice containing YAC HSC7E526 / V12.

Β-galactosidase (LacZ) activity was determined in tissue extracts from control mice and two independent lines expressing the hVPAC2R-HA-lacZ transgene. Enzyme activity was determined using a chemiluminescent reporter system (Glacto-Light Plus, Tropix). ND indicates that no β-galactosidase activity was detected.

For ease of reference, some parts of the text that follow are set forth separately according to the vectors of the first aspect of the invention or the vectors of the second aspect of the invention.

Materials and Methods Insertion Vector Construction of YAC Insertion Vector For genetic manipulation of YAC DNA, we have created a series of modified cassettes that can be inserted into all YAC DNAs.

Cassette containing the lacA reporter gene flanked by pYIV1 IRES and LEU2 selectable markers. This vector can be used for YAC introduced into Leu ^- yeast strain. The details of the fabrication method were as follows.

The 3.5 kb cassette containing the lacZ reporter gene and polyadenylation sequence was separated from pMRβ-lacA-PA (23) by SalI (complete) and EcoRI (partial) digestion, and pBluescript S
K ^- for inserting into EcoRI-SalI sites resulting in pSK-lacA-PA. 1.1 kb XbaI (in the polylinker) -EcoRV (in the lacZ sequence) was converted to a 1.7 kb XbaI-EcoRV fragment from pIRES-bgeo (24) (
By substituting IRES-5'-lacZ) and the IRES pSK ^- introduced into the lacZ-PA, pIRES-
This resulted in lacZ-PA. The 2.2 kb XhoI-SalI fragment containing the LEU2 gene was ligated into pDB248 (2
5) separated from, in the Sall site fits the pIRES-lacZ-PA, was inserted into the same way direction as lacZ gene, pBluescript SK ^- plasmid containing the IRES-lacZ-PA-LEU2 cassette in (PYIV1) (FIG. 1). Five unique restriction sites sandwich the cassette (SacII, NotI and XbaI between T3 primer and IRES, SalI and X between LEU2 gene and T7 primer)
hoI site), allowing insertion of genomic DNA on both sides of the cassette.

PYIV2 Except that the LEU2 gene was replaced by the ADE2 gene so that the plasmid could be directly introduced into the conventional yeast strain (AB1380) used to create most YAC libraries,
V2 is similar to pYIV1. The 2.5 kb Bg / II fragment containing the ADE2 gene was
PBluescr in a direction such that the T7 promoter is adjacent to the 5 'of the ADE2 gene.
The filled HindIII site of ipt SK- was filled in and inserted (pSK-ADE2). IRES-lacZ-
The PA cassette was separated from pIRES-lacZ-PA by digestion with SalI, inserted and then cut with NotI. This fragment pSK ^- ADE2 the EcoRI (filled) and the NotI site insert city, resulting in plasmid (PYIV2) containing IRES-lacZ-ADE2 cassette (Figure 4). Four unique sites (SacII, NotI, SalI and XhoI) are available for cloning.

PYIV3 For many genes, no antibody is available to detect the encoded protein. We introduced a haemaggluttinin (HA) epitope tag into pYIV2 using a commercially available antibody to identify the location of the intracellular YAC transgene protein product. The HA label was introduced as follows.

The translation termination codon of the human VIP2 receptor cloned into the vector pcDNA3 was converted into an XhoI site by PCR-based mutagenesis. XhoI-XbaI site 5'-TC GAG TAC CCA TAC GAT GTT CCA GAT TAC GCC TCC CTC TAG-3 '3'-ATG GGT ATG CTA CAA GGT CTA ATG CGG AGG GAG ATC AGA TC-5' HA epitope The linker encoding the label was replaced by Xh at the end of the VIP2 receptor.
It was cloned into the oI-XbaI site. The receptor-HA fragment was separated from the pcDNA3 vector by BamHI and XbaI (filled) digestion, and into pBluescript SK ⁻ at the BamHI-EcoRV site, where filling removes the XhoI site. Cloning resulted in pSK-VIP2R-HA.

The IRES-lacZ-ADE2 cassette was separated from pYIV2 by NotI (filled) and SalI restriction enzymes, inserted into HindIII (filled) and SalI sites of pSK-VIP2R-HA, and VIP2R- A plasmid containing HA-IRES-lacZ-ADE2 was generated. The HA-IRES-lacZ-ADE2 cassette was separated by Xhol-SalI digestion and inserted into pGEM11Z at the XhoI-SalI site, resulting in pYIV3 (FIG. 3). In pYIV3, the HA-IRES-lacZ-ADE2 cassette is 5 '
It is flanked by NotI and Xhol at the ends and SalI and SfiI sites at the 3 'end to facilitate cloning of genomic fragments for YAC manipulation.

The direction of the pYIV4 ADE gene is opposite to that of pYIV3, two loxP components have been introduced in the same direction, one at the BglII site between lacZ and PA, and one after the ADE2 gene. Other than that, pYIV4 is similar to pYIV3.

[0143] The loxP sequences from pBG, pBluescript at the EcoRI and SalI sites SK ^- was cloned into the (pSK ^- loxP). The ADE2 gene was excised from pSK ^- ADE2 by EcoRI and ClaI (filled) digestion and cloned into pSK ^- loxP at EcoRI and SmaI sites (pSK ^- loxP-ADE2). The EcoRI-SalI loxP fragment was isolated from pSK ^- loxP, blunt-ended with Klenow, and the B between the lacZ and polyA sequences of pSK ^- IRES-lacZ-PA.
Insertion at the glII (filled) site resulted in pSK ^- IRES-lacZ-loxP-PA. IRES-lacZ-l
The oxP-PA cassette is separated by NotI and SalI (filled) and NotI and BamHI (filled).
(Filled in) site and cloned into pSK ^- loxP-ADE2, resulting in the construct pYIV4 (FIG. 4). The pYIV4 vector allows the deletion of the SV40 PA and ADE2 genes in YAC transgenic animals using Cre recombinase, so that the transgene and lacZ reporter gene can be followed by its own 3 'untranslated region. Become.

Preparation of Yeast DNA Yeast DNA was isolated by a combination of the methods of Schedl et al. (26) and Bellis et al. (27). Clones were inoculated into 15 ml of medium (Ura ⁻ / Lys ⁻ ) containing 2% galactose instead of glucose as a carbon source. After 2-4 days, when the cells have grown to late log phase, the plugs are removed and Novozyme digestion is performed for 4-6 hours, as reported by Schedl et al. (28). Next, plug the plug into 50 mM EDTA (2x
(30 min) and use Proteinase K (2 mg / ml) in a buffer containing 0.5 M NaCl, 0.125 M Tris pH 8.0, 0.25 M Na2EDTA, 1% lithium sulfate and 0.5 M β-mercaptoethanol. And digested at 55 ° C. overnight. Next, the plug is washed with TE and
Stored in MEDTA at 4 ° C.

Pulse Field Gel Electrophoresis The DNA plug was washed in TE (3 × 30 minutes), added to a 1% agarose gel, and added to 0.5 × TB
Sealed with 1% agarose in E buffer. The gel was run for 24 hours at 6 V / cm in a 0.5 × TBE buffer at 14 ° C. with a switching time of 60 seconds. After operation, the gel was stained with ethidium bromide and photographed.

Preparation of Amplification Vector pYAM4 pYAM4 was prepared using pYAC4, pBluescript SK ^- and pBG. pBG is pCGS9
At 90 the SalI site was converted to a NotI site, and the PGK-Hyg-loxP cassette
It is a modified version of pCGS990 introduced between the TK genes. pBG is prepared as follows. The following Xhol-NotI-Xhol linker

[0147]

Embedded image

Was used to convert the unique SalI site of pCGS990 into NotI, resulting in pCGS990N. 2044b containing the chloramphenicol resistance gene (cm) flanked by two loxP sites
The pPstI fragment was excised from pYC9lox2cm, blunt-ended with T4 DNA polymerase, and filled with PHA58 containing the hygromycin B resistance gene flanked by the mouse Pgkl (phosphoglycerate kinase 1) promoter and polyadenylation signal.
bound to the hI site. The resulting plasmid (pHA58lox2cm.1) was digested with BglII and
The 3.5 kb BglII fragment containing the oxp-cm-loxP-Hyg cassette was isolated, filled in with Klenow, inserted into the EcoRI site (filled) between the TK and LYS genes of pCGS990N to obtain pCGS990N-Hyglox2cm did. The chloramphenicol resistance gene was removed from pCGS990N-Hyglox2cm using purified Cre recombinase. DNA was purified, transformed into E. Coli XL-1 Blue, chloramphenicol-selected colonies were selected, and pBG was produced.

The 572 bp (SmaI-ClaI) fragment between the cloning site of pYAC4 and CEN4 was
uescript SK ^- was cloned into the SmaI-ClaI site of. Put this fragment in Cla
Excision was performed with I and NotI and inserted into the ClaI-NotI site of pBG, resulting in pYAM3.

The telomere (TEL) 705 bp Xhol-BamHI fragment of pYAC4 was blunt-ended with Klenow and inserted into the SacI-SacII site filled in by the pBluescript SK ^- vector. The direction of TEL in the resulting plasmid (pSK ^- TEL) was confirmed by sequencing using T7 and a reverse primer. pSK ^- TEL is digested with NotI-SalI restriction enzyme and pYAC
3 corresponding regions (pBR322-TEL-TK-LoxP) were replaced to produce pYAC4 (FIG. 6).

Transformation pYAM4 was linearized with NotI. 10m without uracil and tryptophan
l Inoculated in SD medium. Once the yeast has grown to 2 × 10 ⁷ cells / ml, collect it and
Was washed using LTE (0.1 M LiOAc, 10 mM Tris pH 7.5, 1 mM Na ₂ EDTA). 100 μL
Were resuspended in LTE and cells were incubated at 30 ° C. for 1 hour by conventional inversion. 1 μg of linear pYAM4 and 5 μL of carrier DNA (salmon sperm DNA, 10 mg / ml) were added to the cells and mixed. After incubation at 30 ° C. for 30 minutes, 0.7 ml of PEG / LTE (40% PEG3 in LTE)
, 300) was mixed with the cells and incubated at 30 ° C. for 30 minutes. The cells were then heat shocked at 42 ° C. for 5 minutes. The cells were spun at 500 × g for 2 minutes and washed twice with TE. After resuspension in 400 μL TE, cells were cultured in SD medium without uracil and lysine. Plates were kept at 30 ° C. for 3-5 days. Clones that failed to grow on medium without tryptophan were selected for further analysis.

Preparation of Yeast DNA Yeast DNA was isolated by a combination of the methods of Schedl et al. (26) and Bellis et al. (27). Clones were inoculated into 15 ml of medium (Ura ⁻ / Lys ⁻ ) containing 2% galactose instead of glucose as a carbon source. After 2-4 days, when the cells have grown to late log phase, the plugs are removed and a novozyme digestion is performed for 4-6 hours as described by Schedl et al. (28). Next, the plugs are washed in 50 mM EDTA (2 x 30 min) and buffered with 0.5 M NaCl, 0.125 M Tris pH 8.0, 0.25 M Na2EDTA, 1% lithium sulfate and 0.5 M β-mercaptoethanol. Digestion was performed with proteinase K (2 mg / ml) at 55 ° C. overnight. Next, the plug is washed with TE and 0.5M
Stored in EDTA at 4 ° C.

Pulse Field Gel Electrophoresis The DNA plug was washed in TE (3 × 30 minutes), added to a 1% agarose gel, and added to 0.5 × TBE
Sealed with 1% agarose in buffer. The gel was run for 24 hours at 6 V / cm in a 0.5 × TBE buffer at 14 ° C. with a switching time of 60 seconds. After operation, the gel was stained with ethidium bromide and photographed.

Combination of Insertion Vector and Amplification Vector Combination of pYIV3 and pYAM for YAC Transformation Studies Using the vector pYIV3, a hemagglutinin (HA) tag and lacZ reporter gene were
Two YAC clones were introduced into 35D8 (500 kb) and HSC7E526 (630 kb). The latter contains the human serotonin transporter (SERT) and VIP2 receptor (VIPR2) genes, respectively. In order to integrate the lacZ reporter gene into each YAC clone by homologous recombination, the genomic DNA sequence flanking the stop codon of each gene (at least several hundred bp) was inserted into pYI
The V3 vector was introduced on both sides of the HA-IRES-lacZ-Ade2 sequence.

Preparation of pLacZVIPR2 ⁺ Vector The XhoI site in the pBluescript SK ⁻ polylinker was removed by digesting the vector with XhoI and filling the 3 ′ end of the recess with the Klenow fragment of E. coli DNA polymerase I. And yields pSKX. A BamHI-XbaI fragment containing the human VIPR2 cDNA with an HA tag at the C-terminus of the coding sequence (McDonald et al 1997 Bi
ochem Soc 25: 442S) is subcloned from the pcDNA3 vector into pSKX at the EcoRV site such that the 5 'end of the cDNA is adjacent to the T3 primer in the pSKX vector, resulting in pSK-VIPR2-HA. Next, NotI-P of pSK-VIPR2-HA containing VIPR2 cDNA sequence
The stI fragment is replaced by a 1.2 kb NotI-PstI fragment of VIPR2 genomic DNA (PstI section at the last coding exon of the human VIPR gene), resulting in pVHA. IRES-lacA-PA-Ade2 cassette is pI as SalI-NotI (blunted)
Excised from IV2, inserted into the SalI-HindIII (blunted) site of pVHA,
VHAIZA occurred.

By PCR using primers 32366 (5′-CAA ACG GAG ACC TCG GTC CTC GAG CCC CAC-3 ′) and 32496 (5′-CG G GTA CC A AAA TGG TGG GTT GTT CTG TAA-3 ′) 1.6k at the 3 'end of the genomic DNA of the stop codon of the human VIPR2 gene
XhoI and KpnI restriction sites were introduced at both ends of the b fragment. This fragment was subcloned into the XhoI and KpnI sites of the pSK-vector and the p3'VIPR
Yielded 2.

The NotI-SalI fragment of VIPR2, including FIPR2-HA-IRES-lacZ-PA-Ade2, was
Ligation into ho3 digested P3'VIPR2 resulted in the final construct pLacZVIPR2 +. For efficient homologous recombination in yeast, a genomic DN at least several hundred bp long
The A sequence must have the stop codon of the target gene flanked by the HA-IRES-lacZ-Ade2 sequence of pUIV3. In pLacZVIPR2 +, there is a 1.3 kb VIPR2 genomic sequence upstream of the HA-IRES-lacZ-Ade2 cassette and a 1.6 kb VIPR2 genomic sequence downstream.

[0158] PLacZSERT ⁺ vector production pBluescript SK of ^- the polylinker XhoI site at the, the vector was digested with XhoI, removed by filling the 3 'end of the recess with Klenow fragment of E.Coli DNA polymerase I And yields pSKX. A BamHI-XbaI fragment containing the human VIPR2 cDNA with an HA tag at the C-terminus of the coding sequence was
Subcloning into the SKX at the EcoRV site such that the 5 'end of the cDNA is adjacent to the T3 primer in the pSKX vector, resulting in pSK-VIPR2-HA. The 5kb human genomic DNA fragment containing the intron 13 and exon 14 of the SERT gene, PCR primers 3265 (5'ACT GCA TA G CGG CCG C AT CTT TCA TTT GCA TCC CC3 ') and 32853 (5'TGT G CT CGA G AG CAT It was cloned into the NotI-XhoI site of pBluescript SK- using TCA AGC GGA TGT3 ') to generate pIn13. To introduce the HA tag into the SERT gene product, a 5 kb SERT intron 13 sequence (N
The otI-XhoI fragment) was used to replace the NotI-XhoI fragment in pSK-VIPR2-HA, resulting in pIn13-HA. The intron 13 sequence and the HA tag were separated as SacII-ClaI (flattened) fragments, inserted into the SacII and (blunted) NotI sites of YIV2, and pIn13-HA-IZA was inserted. occured. The sequence downstream of the stop codon in exon 14 of the SERT gene was determined using primers 32358 (5 ' CTC CTC GAG AGG AAA AAG GCT TCT 3') and 32359 (5 'TA G GTA CC C TGT TCT CTC CTA CGC AGT TT3'). Was isolated by PCR of human genomic DNA and cloned into the XhoI-KpnI site of pBluescript SK- to generate p3'SERT. Finally, NotI and Sa of pIn13-HA-IZA
The intron 13-HA-IRES-lacZ-Ade2 fragment was separated by lI digestion and p3'SERT
Into NotI and XhoI to generate PlacZSET +.

Introduction of Insertion Constructs into YAC DNA The pLacZVIPR2 + and pLacZSET + constructs were linearized with NotI and introduced into the YAC clones HSC7E526 and 35D8, respectively. Transformants in which the HA-lacZ-Ade2 sequence was incorporated into the YAC DNA by homologous recombination were selected by growing in culture dishes without uracil, tryptophan and adenine. The incorporation of the HA-IRES-lacZ-Ade2 sequence into YAC DNA was confirmed by Southern blotting using an Ade2 probe.

Amplification of Modified YAC DNA The YAC subclone incorporating the HA-lacZ-Ade2 cassette was cloned into NotI-linearized p
Transformation was performed using YAM4. The recombinant material was separated on a selective medium containing no uracil, adenine and lysine, and replica-cultured in a culture dish containing no uracil, adenine and tryptophan. When the YAC left arm (including the TRP1 gene) was successfully replaced by pYAM4, a yeast capable of growing on a medium containing no uracil, adenine, or lysine was obtained, but not on a reverse selective medium.

Tryptophan-sensitive clones were cultured in selective media (Ura ⁻ / Ade ⁻ / Lys ⁻ ) supplemented with galactose instead of glucose as a carbon source. In such a medium, the GAL1 promoter adjacent to CEN4 in the pYAM4 vector is induced. Activation of transcription from the GAL1 promoter interferes with CEN function and causes non-segregation of YAC DNA, resulting in increased YAC DNA per cell.

Transformed Cells / Transgenic Organisms YACs of the invention can be introduced into mammals by appropriate modification of the instructions of Schedl et al (Cited Document 28). By the word "modification" we mean to follow the instructions but utilize the vectors of the invention as appropriate. Obviously, other techniques can be used to transfer the YACs of the invention into mammalian cells, and these other techniques are well documented in the art of the invention (e.g., WO-A -95/14769 and / or Gietz et al 1995 Yeast fol 11 No.4, pp355
-360).

According to Schedl et al (ibid.), Perhaps the simplest method of generating a transformed cell line is spheroblast fusion, a technique disclosed in the previous section of ref. ) Is the import of YAC. However, this method usually results in the integration of the entire yeast genome in addition to YAC, which may obscure the experimental results. In contrast, direct microinjection of DNA into the nuclei of recipient cells allows for purification of YACs before transfer.

The instructions of Schedl et al (ibid) are as follows: Materials 1. SE: 1 M sorbitol, 20 mM EDTA (pH 8.0). 2. TENPA: 10 mM Tn's-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 100 mM NaCl, 30 μM spermine, 70 μM spermidine. 3. IB: 10 mM Tris HCl (pH 7.5), 0.1 mM EDTA (pH 8.0), 100 mM NaCl, 30 μM spermine, 70 μM spermidine. 4. LiDS: 1% lithium sulfate-dodecyl, 100 mM EDTA (pH 8.0) 5. Zymolyase, ICN Biomedicals Inc., Costa Mesa, CA, USA 6. Nusieve low melting point (LMP) agarose, FMC, Rockland, ME, USA 7.Seaplaque low melting point (LMP) agarose, FMC, Rockland, ME, USA 8.Automatic injection system, Zeiss, Germany 9.Femptotips, Eppendorf 10.Injection mold (plug forming substance), Pharmacia, Uppsala, Sweden 11.CHEF- DR 11, pulse field gel electrophoresis (PFGE) system, BIO-RAD Labor
atories, Richmond, CA, USA 12.β-Mercaptoethanol (14M stock solution) [Note: If there is a heavy metal ion in the buffer even in a trace amount, YAC D
Causes decomposition of NA. High quality water must be used for buffer preparation. ]

Methods Preparation of high density agarose plugs for preparative pulse field gel electrophoresis (PFGE) 1. Inoculate 500 ml SD medium (-URA) with yeast clones and grow culture to late log phase. 2. Prepare 1% Seplaque LMP agarose solution in SE buffer containing 14 mM β-mercaptoethanol and store at 45 ° C. until use. 3. Spin down cells at 4000 rpm for 5 minutes (Sorvall RT6000) and resuspend pellet in 50 ml SE. 4. Seal the bottom of the Pharmacia plug former (insert mold) with tape and place on ice. 5. Wash the cells twice with SE (4000 rpm, 5 minutes). 6. After the last washing step, discard the supernatant, wipe the inside of the test tube with a paper towel,
Carefully remove all liquid. The cell pellet should be between 1 and 1.5 ml. 7. Add 200 μL of SE buffer. Attempt to resuspend the pellet using a cut-off tip. The suspension is very thick and difficult to pipette. 8. Transfer a 0.5 ml aliquot of the cell suspension into a 2 ml Eppendorf tube and store at 37 ° C. 9. Immediately before use, dissolve 10 mg Zymolyase in 2.5 ml LMP agarose solution. [Note: Zymolyase does not completely dissolve at this concentration. Weigh the required weight and treat the protein suspension. ]

10. Transfer 0.5 ml of this solution to the yeast cell suspension and pipette in and out using a blue cut-off tip to mix the agarose and cells thoroughly. [Note: Only perfectly homogeneous mixtures yield high quality plugs with even distribution of DNA. Always maintain the solution at 45 ° C. so that the agarose does not solidify. 11. Using a cut-off yellow tip, pipette an aliquot of 80 μL of the mixture into the plug former stored on ice. 12. Transfer into SE buffer containing 14 mM β-mercaptoethanol and 1 mg / ml zymolyase. Incubate at 37 ° C for 4 to 6 hours. 13. Replace the buffer with LiDS buffer using at least 0.5 ml / plug and incubate at 37 ° C with gentle rocking. 14. Thoroughly clean plug in TE at pH until bubbles are no longer visible. Until use, 4 ℃
Save the plug in 0.5M EDTA with. [Note: DNA plugs prepared in this way can be stored without denaturation for at least one year. ]

Separation of Intact YAC DNA for Microinjection 1. Using a comb with several teeth taped, use 0.25 × TAE, 1% agarose to cast a gel and prepare approximately 5 cm. Get the lane. [Note: It is recommended that a preparative lane be created in the center of the gel to ensure that the band is straight. Bands in the preparative lane that are larger than 5 cm may exhibit a "smileing" effect, which may lead to inaccurate DNA excision and thus lower yield / final concentration. If the DNA is concentrated by the second gel operation, standard agarose can be used. Alternatively, use LMP agarose. 2. Wash the high-density plug in pH 8 TE four times for 15 minutes while gently moving on a rocking table. 3. Add plugs next to the preparation lanes. [Note: Best results are achieved using rectangular plugs (such as those produced with Pharmacia plug formers) so that they can be added next to each other without interfering with space. For a small BioRad casting chamber (14 cm x 12.7 cm), use 90 ml of 1% gel. The plug must occupy the entire height of the gel. Therefore, make sure that the comb is in contact with the bottom of the casting chamber when casting the gel. When running the gel, make sure that the casting chamber and PFFGE-chamber are level to avoid DNA loss. Seal the slots with 1% agarose (0.25xTAE).

4. Run PFGE in cooling buffer (0.25 × TAE) using conditions optimized for separating YAC from endogenous chromosomes. [Note: Better segregation from endogenous chromosomes is obtained with a single pulse time than with a gradient over the entire run time. It is useful to test some conditions before starting the separation procedure. ]

5. After running the gel, cut out both marker lanes, including the 0.5 cm preparative lane, and stain in 0.25 × TAE containing 0.5 μg / ml ethidium bromide. Use a sterile scalpel blade to mark the position of the YAC band under UV light.

6. Reassemble the gel and cut out the preparative lane corresponding to YAC-DNA. Y
Cut out the upper and lower slices of AC DNA and save them as marker lanes for the second gel run.

7. Place the gel slice with the YAC slice in the center of the mini gel tray and surround it with 4
Make a mold of% LMP agarose (Nusieve, FMC) gel 0.25xTAE.

Run the gel at 90 ° angle to PFGE in 8.0.25 × TAE (circulation buffer) at 4 V / cm for about 6-8 hours. Run time depends on the size of the gel slice and the agarose used for the PFGE run.

9. Cut and stain two marker lanes to locate DNA. [Note: If the DNA has not yet completely passed through the Nusieve LMP gel, continue electrophoresis. Since it is impossible to digest normal agarose with the enzyme agarase, it is important to cut out only the LMP material. ]

10. Cut out the concentrated DNA from the position corresponding to the YAC DNA lane. 11. Equilibrate gel slice for at least one and a half hours on a rocking platform in 20 ml of TENPA buffer. 12. Transfer the gel slice to a 1.5 ml Ependorf tube and remove any buffer using a fine-tipped pipette. 13. Thaw the agarose at 68 ° C for 3 minutes and centrifuge for 10 seconds to precipitate the molten agarose and incubate at 68 ° C for a further 5 minutes. 14. Transfer test tube to 42 ° C. for 5 minutes. 2 U of agarase (N
ew Englands BioLabs). [Note: Do not add agarose directly from the -20 ° C freezer. Some of the LMP agarose may solidify. The enzyme can be warmed for a few seconds by placing the enzyme in the IRES, melted agarose at the tip of the pipette. Carefully release the enzyme with gentle agitation at the tip. Mixing is achieved by releasing gas bubbles into the solution. Incubate at 42 ° C for 3 hours.

15. Microinjection buffer with floating dialysis membrane (Millipore, pore size 0.05 μM)
The obtained DNA solution is dialyzed against (10 mM Tris: HCl, pH 7.5, 0.1 mM EDTA, 100 mM NaCl, 30 μM spermine, 70 μM spermidine) for 2 hours.

16. Check 0.2 μL of a thin 0.8% agarose gel in a very small slot using a known concentration of λDNA as a standard to determine DNA concentration. [Note: It is common to prepare a 2 ng / μL DNA stock solution. Add this standard solution to 2, 5, 10,
By using 20 ng, YAC DNA can be measured relatively accurately. ｝

By running 17.20 μL of the preparation on a PFGE gel (using a small slotted comb)
Check DNA integrity.

Injection into Cultured Cells The Zeiss Automated Injection System (AIS) can be used to rapidly inject large numbers of cells grown in cell culture dishes. A digital camera attached to the microscope sends images to a computer screen. Next, using an interactive computer program,
A pipette can be placed over the "reference cell" and the tip of the screening needle can be marked. This location is stored by the computer and serves as a reference point for the remaining injections. Other cell nuclei visible on the screen can be marked by clicking them with a computer mouse, and the computer will automatically inject. The amount of DNA injected can be adjusted by the injection home and the pressure set in the Eppendorf injection system. High pressure increases the outflow of the DNA-containing solution. The set pressure depends on the viscosity of the DNA solution and the size of the needle opening, and must be adjusted for each experiment. Standard experimental pressures are between 20 and 150 hectopascals. Confluent culture dishes are most suitable for injection. If the cell density is too low, only a few cells will be injected per frame, but the cells in a dense culture dish will not grow in one plane, making it impossible to inject into all cell nuclei. The efficiency of microinjection depends greatly on the cell type. Best results are obtained using large, easily visible cells with large nuclei.

1. Grow cells to 5% confluence in a 5 cm culture dish in media required by cell type. 2. Immediately before injection, cover the cells with 5 ml of fresh medium and cover the culture dish with 5 ml of liquid paraffin. Preferably, liquid paraffin is used to prevent cell contamination and media evaporation during injection. 3. Computer, microscope, monitor, Eppendorf micro injector,
Switch on the pump and place the culture dish on the stage. 4. Select the command STAGE from the main menu and select the area of the culture dish that is almost dense but still has cells growing on one plane. By clicking, the stage can be moved to two intersecting arrows. The direction blade of the arrow indicates the direction in which the stage moves. The distance from the center of the intersection determines the speed at which the stage moves. 5. Return to the main menu and select MARK / INJECT. A new menu will appear, giving you the following options:

STORE DATA: A file can be created that stores the location of the injected cells. To use this option, a mark must be made on the bottom of the culture dish to mark the instrument with left and right reference indicators (cross-cuts on both sides).
After placing the culture dish on the stage, locate the mark and click the cursor in the appropriate box to record the reference index. When creating a file, the operator name and sample name must be entered.

APPEND: You can go back to the previous file to find the microinjected cells. NO FILE: This option does not record the cells injected and is sufficient for many applications.

6. Select the number of the frame you want to inject by filling in numbers less than 10 for X and Y values. The frame is the window seen on the screen, and thus represents a place in which cells can be simultaneously marked and injected. Each frame has its own X and Y coordinates. The computer first moves along the X axis. Five
The x10 frame arrangement allows for the injection of more than 1000 cells, depending on the density of the culture dish.

7. Click DATA OK. 8. Put 1.5 μL of the DNA solution into the Eppendorf microloader and insert it into the Eppendorf femput tip located at the bottom of the tip. Release the DNA solution slowly so that air bubbles that can block the needle do not enter. 9. Attach the tip to the needle by twisting the tip off the cover and screwing it into the injection needle of the microscope. 10. Select an adjustment option from the menu. Using the mouse, lower the needle by clicking on the arrow in the center of the screen. The moving speed is determined by the distance from the center. When approaching the surface of the culture dish, start at a high speed and slow down. Once the needle contacts the medium, look at low magnification and use the micrometer screw on the pipette holder to center the needle on the frame. Change the lens to high magnification. Focus on the plane midway between the cell and the needle and lower the needle into focus. Repeat this procedure until the tip of the needle pushes the cell down. This creates a small loop around the tip of the needle when the cells are depressed. 11. The following options are available to adjust the position of the needle.

MARK TIP: A reference point can be set for computer software. To adjust, click on the very tip of the injection needle. INJECTION TIME: Determines the time the needle remains in the cell and is therefore a parameter of the volume injected into the nucleus. This time must fluctuate with pressure, tip, size, etc. 0.2 seconds is a good value to start. MOVE STAGE: The stage can be moved by the mouse. RESTART: Return to the main menu and reset any parameters. HOME: Return the needle to its original position. POSITION OK: Click this when you are ready to start the injection.

12. Click MARK NEXT to perform the injection. This can instruct the computer to inject the nucleus of the cell. Click MARK and continue to the nucleus. Click INJECT to start the injection. The computer performs the injection on the marked cells. Successfully injected cells can be identified by a temporary dramatic edema of the nucleus. If the cells do not change after multiple injections, check for the following possibilities:

Injection needle is occluded: Eject DNA using high pressure button (P) on injection device.
If that doesn't work, you need to change the needle. Computer is injecting on the wrong side: press the yellow button to stop the injection and increase the needle one step down or up. Take care not to lower the needle too much on the surface of the culture dish. Pressure too low: Increase P1 pressure. Note that too high a pressure may cause cell rupture.

Press the mouse button whenever adjusting the height of the needle during injection, or press RESTART again, taking care that the needle tip is filling the entire frame. Alternatively, it can be run with CONTINUE.

13. To end the injection, press RESTART, MARKINJECT, HOME, EXIT.

Injection of Pronuclei into Mouse Fertilized Oocytes Transgenic mouse procedures include the isolation of fertilized oocytes from superovulated females, microinjection of DNA into the pronucleus, injection of injected oocytes. False pregnancy Includes transfer to Foster Mother. A detailed description of these steps can be found, for example, in
Hogan, Murphy and Carter (1993 Transgenesis in the mouse in “Transgenes
is Techiniques ”, Methods in Molecular Biology vol. 18 Ed.Murphy and Car
ter, pp 109-176.Humana Press, Totowa, New Jersey) and Reference 28 (subsequent chapters)-each of which is incorporated herein by reference. And

Preparation of DNA constructs for injection usually involves a filtration step in which the DNA passes through a membrane having a pore size of 0.2 μM. This step is recommended to prevent blockage of the injection needle by dust particles in the DNA solution. YAC DNA preparations should not be filtered at this stage due to shear forces. We have found that needle occlusion is relatively rare, even with successful agarose digestion. In some cases, it is preferable to centrifuge the DNA for 5 minutes to remove undigested gel pieces. However, it is strongly recommended that the DNA concentration be measured after the centrifugation step, since small particles of agarose may capture the DNA.

Some of the DNA preparations are very sticky, probably due to incomplete agarose digestion. In such a case, the lysis of the injected oocytes is seen at a higher rate, and the injection needle must be changed more frequently. On the next injection day, care must be taken to prepare a new batch of DNA and digest all of the agarose. Avoid contact with the pronucleus during injection more than with normal constructs. Once in contact, the pronucleus attaches to the needle and is lifted. If this happens, replace the microinjection pipette immediately. The percentage of lysed oocytes should not be too high compared to the normal construct. The injected oocytes can be transferred to the oviduct of a pseudopregnant Foster mother on the same day, or can be incubated overnight at 37 ° C. in M16 buffer. 10ng /
A concentration of μL of DNA should provide the normal viability (20-30%) of the transferred embryos.

Transgenic animals can be identified by PCR or Southern blot analysis using DNA isolated from the tip of the tail. With a 250 kb construct, about 10 to 2
0% of the offspring should have YAC DNA integrated. Once a transgenic system has been established, it is important to confirm the integrity of the integrated construct. This can be achieved by conventional PFGE mapping using several probes scattered on the YAC, but requires detailed knowledge of the restriction map of the construct.
Alternatively, the RecAhou method can be used to dissociate the entire YAC from the mouse genome.

Results Insertion Vectors General Analysis of genes expressed in YACs in transgenic animals can be performed very easily by utilizing a reporter gene to accurately and sensitively detect transcription sites in cells. We have created a series of YAC-modified vectors (pYIV1, pYIV2, pYIV3, pYIV4) that can be inserted into YAC after the translation start or stop codon. A common feature of all of these vectors is that they include a lacZ reporter gene downstream of the viral internal ribosome entry site (IRES), along with a selectable marker. Since the lacZ reporter gene is expressed in the same pattern as the transgene in transgenic animals expressing these constructs, simple histochemical staining can be used to analyze the expression, regulation and function of the transgene. it can. In this way, in
It provides a more complete picture of the expression pattern of the transgene than standard methods such as situ hybridization. The pYIV1 vector can be used for YAC introduced into the Leu ^- yeast strain, and pYIV3, pYIV3 and pYIV4 can be directly introduced into the yeast strain (AB1380) used for the construction of most YAC libraries. One of the vectors (PYIV
3) is capable of fusing the HA epitope tag sequence (derived from influenza hemagglutinin) to the carboxyl terminus of the expression product of the gene of interest, and
The protein product of the transgene can be detected using the 12CA5 monoclonal antibody. pYIV4 contains the loxP component flanking the SV40 polyadenylation signal and the ADE2 gene. In transgenic animals produced using pYIV4, the polyA sequence and the ADE2 gene sequence can be deleted using Cre recombinase, so the transgene and lacZ reporter gene are -Sandwiched by untranslated regions. Comparison of animals containing the SV40 polyadenylation signal and the ADE2 gene with those with these sequences deleted reveals the function of the 3 'sequence of the transgene.

Expression of IRES-lacZ from pYIV1 in YAC Transgenic Mice The expression of the LacZ reporter gene was examined in transgenic mice expressing a YAC clone modified using the pYIV1 insertion vector. Insulin-like growth factor II (IGF2) cDNA was transferred to the Wilms tumor (wt1) gene promoter (480 kb human YA).
(Isolated from C clone). Next, the promoter and cDNA were ligated with pYIV1
Was inserted into the 5 'NotI-XbaI site of the IRES-lacZ-LEU2 cassette. 3 'of the LEU2 gene
The first intron of the wt1 gene was cloned. This construct was introduced into a 480 kb YAC by homologous recombination. After amplification, the modified YAC DNA was separated and microinjected into fertilized eggs. Eight lines of transgenic mice were generated, five of which expressed the lacZ reporter gene. All of the expressed systems showed the same X-Gal (X-Gal, β-
It is clear that Gal and lacZ are synonyms. ) A staining pattern (FIG. 5) resulted. These data demonstrated that the IRES-lacZ reporter gene functions in the YAC insertion vector.

Amplification Vector Improved Homogenous Recombination Efficiency of pYAM4 To evaluate the efficiency of homologous recombination of pYAM4, plasmids were linearized with NotI and diversified from ICI, ICRF and chromosome-7-specific YAC libraries. Inserted into YAC clone. Recombinants were isolated on a selective medium without uracil and lysine and replica plated on culture dishes without uracil and tryptophan. by pYAM4
Successful replacement of the YAC left arm (including the TRP1 gene) yielded yeast that could grow on media without uracil, adenine, and lysine, but not on the reverse selection media.

Out of a total of 1266 Lys ⁺ clones analyzed, 167 clones failed to grow on medium without tryptophan (Table 1). That is, homologous recombination caused the deletion of the TRP1 gene in 167 clones. The retrofitting efficiency of pYAM4 is 13.3% overall, which is significantly higher than pCCGS990 and pCGS966 (0.5-2.5%).

[0197]

[Table 1]

The increase in retrofitting efficiency is probably due to the 572 bp from pYAC4 adjacent to CEN4.
This is probably due to the introduction of the ClaI-SmaI fragment. When the SmaI-ClaI fragment of pYAM4 was deleted by NotI-ClaI digestion or when pYAM4 was linearized by ClaI, the frequency of tryptophan-sensitive clones was not significantly different from that obtained with pCGS990. .

Amplification of YAC DNA with pYAM4 Tryptophan-sensitive clones were cultured in a selective medium (Ura- / Ade- / Lys-) supplemented with galactose instead of glucose as a carbon source. In such a medium, the GAL1 promoter adjacent to CEN4 in the pYAM4 vector is induced. Activation of transcription from the GAL1 promoter interferes with CEN function and causes non-segregation of YAC DNA, resulting in increased YAC DNA per cell.

As shown in FIG. 7, human YAC DNA was amplified 3- to 5-fold depending on the size and nature of the insert. Amplification is not as high as achieved by pCGS990, but is useful for separating YAC DNA that is more enriched for transgenics. Amplification may be further improved by the introduction of additional conditional promoters, such as ADH2, adjacent to CEN4, or additional selection genes.

We introduced the bacterial hygromycin B resistance gene into pYAM4 under the control of the mouse Pgk1 promoter. YAC DN isolated after retrofitting with pYAM4
A can be introduced into mammalian cells, such as embryonic stem (ES) cells, which is an alternative to microinjection of YAC DNA to produce YAC transgenic animals (29).

If a genomic fragment (present in YAC) is cloned into the ClaI-NotI site of pYAM4, cleavage of large YAC and amplification of truncated YAC DNA can be achieved in one step.

Combination of Insertion Vector and Amplification Vector The results are shown in FIGS. In this regard, FIGS. 8 and 9 are photographs of the gel. Amplification of YAC DNA is observed in FIGS. In this regard, lane 1 is unamplified YAC DNA and is present in the original 35D8 YAC clone, lane 2 is unamplified YAC DNA in another YAC clone containing the SERT gene (132C6), and lane 3 is 35D8
/ Amplified YAC DNA in DD6. The blot was hybridized with genomic probes downstream (FIG. 8) and upstream (FIG. 9) of the SERT gene.

Amplification and Purification of YAC DNA Incorporation of the amplification vector pYAM4 into YAC clone 35D8 and HSC7E526 greatly increases the yield of YAC DNA obtained. These results are shown in FIG. In this regard, FIG. 11 is a photograph of a gel prepared by Southern blotting and hybridized with a ³² P-labeled pBR322 probe to detect YAC hybridization. In each lane, the hybridization band corresponding to YAC DNA is indicated by an arrow. Lane 1 is yeast-derived DNA containing YAC clone 35D8. Lane 2 is yeast-derived DNA containing YAC clone 35D8 / D6, where YAC is the amplification vector p.
It has been modified by the integration of YAM4 and the insertion vector pYIV3. Lane 3 is YAC DNA isolated from clone 35D8 / D6 before microinjection. lane
4 is yeast-derived DNA containing the YAC clone HSC7E526 / V12, and YAC is the amplification vector pYAM
4 and modified by integration of the insertion vector pYIV3. Lane 5 is YAC DNA isolated from clone HSC7E526 / V12 before microinjection.

Generation of YAC Transgenic Mice Modified YAC DNA was excised from a 1% pulsed field agarose gel in 0.25 × TAE buffer and concentrated in 4% low melting point agarose. Gel slices containing YAC DNA were equilibrated with microinjection buffer (TE pH 7.0 with 0.1 M NaCl) and digested with gelase. Prior to injection into fertilized eggs, YAC DNA was dialyzed against microinjection buffer for 2 hours.

298 fertilized eggs were injected with 35D8 / D6YAC DNA and 364 fertilized eggs were injected with HSC7E526 / V12. After transfer of the injected oocytes into the oviduct of pseudopregnant female mice, a total of 190 mice (28.7%) were born, of which 26 (13.7%) were measured by PCR, as shown below. It had YAC DNA.

[0207]

[Table 2]

PCR Measurement of the Size of the Integrated Construct A series of PCR primer pairs spanning the two YAC vector arms and the SERT (B to C: Table III) and VIPR2 (I to L: Table III) genes, respectively. For detection, the size of the integrated YAC35D8 / D6YAC and HSC7E526 / V12 constructs in each transgenic primary animal was determined using two sets of PCR primers (A and H: Table III).

[0209]

[Table 3]

The results are shown in FIGS. In this regard, FIG. 12 shows the size of integrated YAC DNA in transgenic primary animals with 35D8 / D6. FIG. 13 shows integrated YACs in transgenic primary animals with HSC7E526 / V12.
Indicates the size of DNA. For each primary animal, the possible range of the transgene as determined by PCR is indicated by a black bar and a light circle indicates the presence of the marker. The location of these markers is shown in the schematic diagram of the 35D8 / D6 YAC construct (FIG. 12) and the HSC7E526 / V12 YAC construct (FIG. 13), drawn from the above markers.

Two independent transgenic primary mice with intact YAC35D8 / D6 (A102.3
A102.5, A105, Fig. 12) and six independent transgenic primary mice (A108, A108.1, A108.2, A108.3, A110, Fig. 13) with intact YACHSC7E526 / V12 were identified. Was done. Therefore, a beneficial number of mice born in this study carried intact YAC DNA.

Germline Transmission An important feature of pYAM4 is that thymidine kinase (
It is not necessary to include the TK) gene. When TK gene is not present, pYA
The YAC transgene was found to be transmitted from both primary males and females to the next generation, as determined by primer pair A obtained from the hygromycin resistance gene in M4. Therefore, preferably, the TK gene is not present in the vector of the present invention.

Β-Galactosidase Immunocytochemistry Animals were perfused from the heart with 4% paraformaldehyde in 0.1 M PBS. Brains were post-fixed overnight in the same solution, then transferred into PBS the next day and stored in that solution until sectioning. Brains were infiltrated with 30% sucrose overnight, and then 25 μM thick sections were cut with a cryostat. Sections were collected in multiwell plates containing PBS and then processed for immunocytochemical studies. Sections are 0.1% Triton X-1
Treated with 00 and 0.02% H ₂ O ₂ solution for 30 minutes, then rinsed twice with PBS for 5 minutes. This is followed by a further 30 minute blocking phase with a 2% solution of normal donkey serum. Sections were incubated for 48 hours in a primary anti-β-galactosidase antibody (5′Prime3′Prime, Inc., diluted 1: 20000). Using a biotinylated donkey anti-rabbit IgG (diluted 1: 1000 from Jackson, 60 min), ABC Elite Kit (diluted 1: 1000, 60 min from Vector) for 60 min, and using NiDAB as a chromophore, immunoreactive sites Was made visible.

LacZ Staining of Mature Transgenic Mice Expressing YAC Construct HSC7E526 / V12 Mice are anesthetized with a lethal dose of sodium pentobarbital and briefly perfused from the heart with 0.9% sodium chloride to remove blood. This was followed by longer perfusion with ice-cold fixative (4% paraformaldehyde, pH 7, dissolved in 0.1 M sodium phosphate buffer). After perfusion with about 150 to 200 ml of fixative, the brain and visceral organs were immediately removed and fixed with the same fixative at 4 ° C for 2 to 4 hours. Subsequently, a 2 to 5 mm coronal section of the brain was cut out, and the brain and other organs were washed with phosphate buffered saline (PBS) containing 2 mM magnesium chloride, 0.01% sodium deoxycholate, and 0.02% NP40, pH 7. Washed twice at 4 in RT. After washing, all tissues were treated with 1 mg / mL X-Gal, 5 mM potassium ferricyanide, 2 mM magnesium chloride, 0.01 mM in 5 mM potassium ferricyanide.
Transfer to a solution containing% sodium deoxycholate and 0.02% NP40 in PBS, pH 7.4 and incubate at 30 ° C overnight. After staining, tissues were washed in PBS, cleared in 40% and 80% glycerol (v / v) in PBS and photographed.

The distribution of β-galactosidase activity in transgenic mice was determined by the published distribution of VIPR2 (Cagampang et al., 1998; Inagaki et al., 1994; Shew
ard et al., 1995; Usdin et al., 1994) and selected VIP2 receptor agonists Ro25-1553
(Vertongen et al., 1994). In particular, high levels of β-galactosidase expression were detected in (i) the suprachiasmatic nucleus. Here, VIP and / or PACAP, acting via the VIP2 receptor, may play an important role in regulating circadian variability. VIP immunoreactivity (Takahashi et al., 1989), prepro VIP mRNA (Albers et al., 1990; Gla
zer and Gozes, 1994; Gozes et al., 1989) and VIP2 receptor mRNA (Cag
ampang et al., 1998) and VIP2 (Piggins et al., 1995) have diurnal
P antagonists (Gozes et al., 1995) and VIP2 antisense oligodeoxynucleotides (Scarbrough et al., 1996) have been shown to impair circadian function. (ii) In the pancreas (FIG. 5c). Here, VIP and PACAP stimulate insulin release in β-cells by interacting with the VIP2 receptor.

Chemiluminescence analysis of β-galactosidase in mouse tissues using the Tropix Glacto-Light Plus kit Tissues were dissected from mice, frozen on dry ice and stored at -70 ° C. Thaw this and add 100-400 μL of cold lysis buffer (supplied with the kit, just before use).
Add 0.2 mM PMSF and 5 μg / ml leupeptin. ) Was immediately homogenized. After homogenization, the samples were centrifuged at 12000 g for 10 minutes at 4 ° C. Aliquots of the supernatant were stored at -70 ° C for protein concentration measurements and the rest were incubated at 48 ° C for 60 minutes to inactivate endogenous β-galactosidase. After centrifugation at room temperature for 5 minutes, 20 μL of each sample was used for analysis. 200 μL of Glacto-Lig
ht Add reaction buffer and incubate at room temperature for 60 minutes, then 300 μL of Acce
An lerator was added and the TD-20 / 20 samples were counted on a luminometer (20 seconds after a 5 second delay). Determine protein concentration using the Bio-Rad assay and measure activity in light units
Expressed as / min / mg protein.

Discussion Insertion Vectors Almost all YAC transgenic animals reported to date have been made with unmodified YAC DNA. Transgene expression in these animals can only be assessed by in situ hybridization, Northern blot analysis, PCR and / or immunohistochemistry. By introducing a reporter gene into YAC DNA, the method for detecting the expression of the introduced gene can be simplified.

We have created a series of YAC modified vectors (pYIV1, pYIV2, pYIV3, pYIV4) that can be inserted into YAC after the translation start or stop codon. Since these vectors contain the lacZ reporter gene downstream of the internal ribosome entry site (IRES) of the virus, simple histochemical staining can be used to analyze transgene expression, regulation and function. This method provides a more complete picture of the expression pattern of the transgene than standard methods such as in situ hybridization. One of the vectors (
(PYIV3) can fuse the HA epitope tag sequence (derived from influenza hemagglutinin) with the carboxyl terminus of the expression product of the gene of interest, thus detecting the protein product of the transgene using a commercially available 12CA5 monoclonal antibody can do. pYIV4 is a lox that sandwiches the SV40 polyadenylation signal and the ADE2 gene.
Contains P component. In transgenic animals produced using pYIV4, the polyA sequence and ADE2 gene sequence can be deleted using Cre recombinase, so that the transgene and lacZ reporter gene are '-Flanked by untranslated regions.

Thus, in summary, transgenic technology has played an important role in understanding gene function and regulation in vivo, and in generating animal models of human genetic diseases. The development of Yeast Artificial Chromosome (YAC) technology has enabled the cloning of DNA fragments of several thousand kilobases in size between two YAC vector arms, with all genes and components necessary for faithful regulation. Most (if not all) of the transgenic animals became easier to transfer.

[0220] Analysis of genes expressed in YACs in transgenic animals can be greatly facilitated by using the reporter genes of the present invention to accurately and sensitively detect transcription sites in cells.

Amplification Vector The Importance of YAC Technology and YAC Transgenic The development of yeast artificial chromosome (YAC) technology has enabled the cloning of DNA fragments of thousands of kilobases in size between two YAC vector arms. . Using YAC,
It is possible to clone the complete sequence of a large gene or gene complex that exceeds the cloning limit in conventional bacterial cloning vectors such as plasmids (10 kb), bacteriophages (15 kb), cosmids (50 kb). The cloning of such large DNA fragments is essential for physical genomic mapping (1) and isolation of large genes associated with human genetic diseases (2,3). Bacterial artificial chromosome (BAC) (4) and P1 artificial chromosome vector (5) have high cloning ability (200kb)
Gene manipulation in these vectors is relatively difficult. Because of the high homologous recombination efficiency in yeast, gene manipulation in YAC vectors can be easily performed.

[0222] Transgenic technology is useful in understanding gene function and regulation in vivo.
It has also played an important role in creating animal models of human genetic diseases. It is well recognized that transgenes, including genomic DNA along with introns and essential regulatory sequences, are better expressed in vivo than cDNA-based constructs (6-10).
Utilization of the YAC construct to produce transgenic animals facilitates the presence and transfer of most (if not all) components required for faithful regulation of the gene, resulting in low levels of the gene in the transgenic animal. Positional effects associated with the integration site, which can cause expression and possibly abnormal patterns, can be avoided. However, the efficiency of producing transgenic animals when using YAC DNA is lower (1-5%) than when using conventional vectors.

Importance of YAC Amplification The YAC vector (eg, pYAC4) contains a yeast centromere, two telomeres, and two selectable markers (TRP1 and URA3). After integration of the YAC DNA, the yeast can grow on media without uracil and tryptophan. In these selection conditions, YAC cloning is replicated along with the endogenous host chromosome, producing only one copy of YAC DNA per cell. Using standard protocols, YAC DNA at a concentration of 1 μg / ml can be separated. However, if the YAC centromere is inactivated by transcription induced from the GAL1 or ADH promoter, the copy number will increase significantly. This increase is due to the YA
This is considered to reflect the bias of C separation and the loss of daughter cells without YAC from the population under the selection conditions.

The lower efficiency of transgenic animals made using YAC DNA compared to DNA (about 10%) made from conventional vectors (about 10%) is likely due to the availability of YAC DNA available for injection. At low concentrations. Also, conventional YACs replicate one copy per cell in yeast.

Therefore, assuming that 2 pl of 500 kb YAC at a concentration of 1 ng / μl is injected into the pronucleus, only one molecule of YAC DNA is introduced into a fertilized egg. Therefore, amplification of yeast artificial chromosomes in yeast should provide a method for isolating higher concentrations of YAC DNA that enhances the success rate of production of YAC transgenic animals (ie, transgenic mammals containing YAC).

To date, two vectors, pCGS96, are available to amplify YAC DNA.
6 (11) and pCGS990 (2) have been reported. Both vectors contain the conditional centromere and the heterologous (herpes simplex virus) thymidine kinase (TK) gene. YAC DNA smaller than 600 kb is efficiently amplified (3 to 11 copies / cell). Recently, pCGS966 has
Many new YAC libraries have been constructed (13-15). However, this vector
When used for YAC modification (retrofitting), the frequency of left arm substitution is very low (0.5-2.5%) (12). Most importantly, expression of the TK gene in the testes of transgenic mice impairs spermatogenesis and causes male infertility (16-22). Because of this complication, these vectors are unsuitable for transgenic studies.

Features of the New YAC Amplification Vector pYAM4 We have created a YAC amplification vector that has a number of advantages: 1) Amplify YAC DNA 3-5 fold. 2) Unlike existing vectors, it does not contain the herpes simplex virus thymidine kinase (TK) gene, which causes male infertility in transgenic mice. 3) The homologous recombination efficiency is much higher than the existing YAC amplification vector (13%). 4) Includes a selectable marker (hygromycin B resistance) that facilitates the transfer of YAC DNA into embryonic stem cells and other cell lines. 5) Can be used for targeted deletion of sequences cloned in YAC vectors. 6) Substances that may influence the NOI expression site / region and the NOI expression pattern by fusing NOI (for example, SERT gene) with a reporter gene (for example, LacZ). Testing becomes easier. 7) Transgenic mice that overexpress the human VIPR2 gene together with β-galactosidase facilitate the development of substances that can affect VIP2 activity in humans.

[0228] Two classes of substances can be identified. (i) a substance that modulates human VIP2 receptor expression, which is identified by its ability to affect β-galactosidase activity in transgenic mice; and (ii) a human VIP2 receptor agonist and antagonist, Therefore, an animal model is provided by a transgenic mouse in which the human VIPR2 gene is expressed. Optimally, a YAC transgenic animal, such as a mouse lacking the VIP2 receptor (`` knockout ''), produces a `` humanized '' animal that displays the same pharmacology as the VIP2 receptor is observed in humans May be bred from a point of view.

By way of example, substances acting in the suprachiasmatic nucleus that can affect VIP2 receptor activity can prove useful in treating disorders of circadian dysfunction. Such impairments can impair physical and mental health; (i) extreme conditions in work patterns (shift work); (ii) staggered travel (jet fatigue); and (iii) At age, (iv) can occur in dementia. Such substances are
It may also prove useful in treating sleep disorders, seasonal emotional disorders (SAD), eating disorders and premenstrual syndrome.

As a further example, substances that act in the pancreas that act as human VIP2 receptors or agonists and that can modulate the expression of receptor antagonists may be useful in the treatment of diabetes .

In summary, introduction of the lacZ reporter gene by utilizing pYIV
Along with the amplification of YAC DNA by use of 4, the production of YAC transgenic animals and the analysis of these animals from the viewpoint of expression, regulation, and function of genes present in YAC DNA are significantly easier. Should be.

Overview In these studies, we investigated a series of YAC-modified vectors (eg, pYIV1) containing a reporter gene (eg, the lacZ reporter gene) to facilitate studies of tissue distribution and regulation of the YAC transgene. , PYIV2, pYIV3, pYIV4). These vectors have the potential to find wide application in transgenic research.

In these studies, we also show the generation of a YAC amplified vector (eg, pYAM4) that has many advantages over conventional vectors and is suitable for amplifying YAC DNA to generate transgenic mice. are doing.

In these studies, we used the modified vectors (eg, pYIV1, pYIV2, pYIV1,
A combination of YIV3, pYIV4) with the above YAC amplification vector (eg, pYAM4) is also shown. This combination of these vectors has the potential to find wide application in transgenic research.

For example, the vector of the present invention, particularly the insertion vector, can be used to transfer other artificial chromosomes (ie,
Other artificial chromosomes), which in turn can be used to prepare transgenic organisms (including animals). In this alternative embodiment, the above statements relating to the invention and description still apply, in which case YAC refers to all suitable artificial chromosomes, preferably to artificial yeast chromosomes.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and alterations of the described method and apparatus of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be apparent that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology and related fields are
It is intended to be within the scope of the following claims.

References

[Table 4]

[0238]

[Table 5]

[0239]

[Table 6]

[0240]

[Table 7]

[0241]

[Sequence list]

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 2, 2000 (200.2.2)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (31) Priority claim number 9806072.6 (32) Priority date March 20, 1998 (March 20, 1998) (33) Priority claim country United Kingdom (GB) (31) Priority claim number 9824275.3 (32) Priority date November 5, 1998 (11.5 November 1998) (33) Priority claim country United Kingdom (GB) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Harmer, Anthony John HK, UK 166 NA Edinburgh, Riverton Place 56 F term (reference) 4B024 AA11 AA20 CA01 DA02 EA04 FA10 FA13 HA17

Claims (32)

[Claims] 1. A YAC vector containing IRES. 2. A YAC vector whose reporter gene expression product contains a reporter gene capable of generating a visually detectable signal. 3. A YAC vector whose reporter gene expression product comprises a reporter gene capable of generating an immunologically detectable signal. 4. A YAC vector containing an IRES and a reporter gene, wherein the expression product of the reporter gene can generate a visually detectable signal. 5. A YAC vector comprising an IRES and a reporter gene, wherein the expression product of the reporter gene can generate an immunologically detectable signal. 6. pYIV1. 7. pYIV2. 8. The pYIV3. 9. pYIV4. 10. A YAC vector or YAC containing a selection gene that can be specifically removed from the YAC vector or YAC. 11. A YAC vector comprising a nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4, or a sequence shown as a variant, homolog or derivative thereof. 12. A YAC or vector capable of modifying a YAC vector, comprising a nucleotide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 4, or a sequence shown as a mutant, homolog or derivative thereof. 13. The pYAM4. 14. YAC prepared by one or more vectors according to any of claims 1 to 13. 15. Use of an IRES to modify a YAC vector or YAC. 16. Use of a nucleotide sequence comprising the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4, or a variant, homolog or derivative thereof in the vector to prepare a YAC vector or YAC. 17. A sequence shown as SEQ ID NO: 1 or SEQ ID NO: 4, or a variant, homolog or derivative thereof in a vector to increase the expression efficiency of one or more NOIs within a YAC vector or YAC. Use of a nucleotide sequence containing 18. The NOI expression pattern is determined by measuring a detectable signal (such as a visually or immunologically detectable signal) generated by an expression product of the reporter gene. A YAC transgenic mammal that simultaneously expresses a NOI and a reporter gene. 19. A YAC transgenic mammal that expresses a reporter gene under the control of a human NOI regulatory sequence. 20. A YA for testing the potential of pharmaceuticals and / or veterinary medicines.
Use of C transgenic mammals. 21. The step of administering a substance to a YAC transgenic mammal as defined in claim 18, wherein said substance modifies a NOI or expression product (to affect expression pattern or activity). Determining the presence or absence of a NOI expression pattern or the activity of the expression product thereof. 22. Screening for a substance useful for treating any one of disorders of circadian function, sleep disorder, eating disorder, premenstrual syndrome, autoimmune disease, birth defects and / or sexual dysfunction in women. 22. The analysis method according to claim 21 for: 23. A substance identified by the method according to claim 22. 24. (a) a step of performing the analysis method according to claim 21 or 22, and (b) identifying one or more substances that affect the expression pattern of NOI or the activity of the expression product thereof. And (c) preparing the one or more identified substances in large quantities. 25. (a) performing the analysis method according to claim 21 or 22; and (b) identifying one or more substances that affect the expression pattern of NOI or the activity of the expression product thereof. And (c) preparing a pharmaceutical composition comprising one or more identified substances. 26. (a) a step of performing the analysis method according to claim 21 or 22, and (b) one or more substances that influence the NOI expression pattern or the activity of the expression product. And c) modifying one or more identified substances that cause different effects on the NOI expression pattern or the activity of the expression product. 27. The YAC according to claim 2, claim 3, or claim 4 or claim 5, for screening for a substance that may affect the expression pattern of NOI or the activity of the expression product thereof. , Or Claims 6 to 9 or Claim 13 (
(Including combinations thereof). 28. An in vitro method for preparing a pharmaceutical composition for treating a disease or condition associated with the expression pattern of NOI or the activity of the expression product thereof, according to the method of claim 21 or 22. Use of a substance that affects the activity of the NOI expression pattern or its expression product when analyzed in step 2. 29. Use of a substance identified by the analysis method according to claim 21 or 22 during the manufacture of a medicament that affects the expression pattern of NOI or the activity of the expression product thereof. 30. The analysis method according to claim 21 or claim 22, wherein
A substance that affects the expression pattern of NOI or the activity of its expression product when analyzed by itro, and is a pharmaceutical composition for treating a disease or condition related to the expression pattern of NOI or the activity of its expression product Use of substances in the preparation of 31. Use of a substance identified by the analysis method according to claim 21 or 22 in the manufacture of a medicament that affects the expression pattern of NOI or the activity of an expression product thereof. 32. A vector substantially as described herein and associated with the accompanying drawings.