JPH06508756A - Intervening deletions in chromosomal DNA - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Intervening deletions in chromosomal DNA JF, anti-old branch The field of the invention is directed nodificative modifications of chromosomal DNA. ati.

n).

The large size of mammalian chromosomes makes it difficult to determine the organization of the chromosomes. It's a difficult job. The Human Genome Program, which has already been launched, An attempt has been made to sequence the entire genome. Part of this program is Towards using the genome of other eukaryotes with simple genomes, e.g. It is being

Numerous paths have been taken in an attempt to determine the relationship between the human genome and structural gene locations. Tools have been developed and developed to assist in twenty major tasks. It will probably continue.

In addition to my interest in elucidating the structure of mammalian chromosomes, I am also interested in manipulating chromosomes and There is also substantial interest in providing techniques for modifying homologous recombination There is much interest in (HR), which suggests that DNA at a given site allows modification of These modifications include a wide variety of changes, such as insertions, deletions, Can be any of the permutations, and combinations thereof. therefore , attaching a specific gene to prevent its expression through homologous recombination and the gene can be mutated, e.g., to provide expression of the wild-type product. A constitutive promoter can be modified to a regulated promoter by substitution. DNA can be inserted or exchanged to replace the It is possible to introduce an enhancer adjacent to a structural gene to enhance expression in offspring. can.

Furthermore, the frequency of homologous recombination in mammalian cells is highly variable and often unspecified. have been observed to depend on specific loci in the cell type. Therefore, the target Why might it be useful to change one of the two homologous ends of the construct? If so, repeat to allow that target sequence to search for recombinant genes. This is because the introduction of the element is actually performed.

These various modifications and others modify cells for expression of desired products. cells for use as therapeutic agents, potentially successful drugs, etc. For modification, it can be used for gene therapy. Therefore, chromosome To provide a technology in which controlled modification of There is a real interest in

1 more pattern indigo Introduction of YaCs into mammalian cells was described by Pavan et al., (1990). Mo1. Ce1l.

Same sex, Larman et al., (1983) 1 bid 80:396 6-3970 and Adams et al., (1980) Nucleic Aclds Res, 8:6113-6128.

Thomas and Capecchi (1987) Ce11.51:50 3-512 is a site-specific mutation using gene targeting in mouse embryo-derived cells. Describes natural mutagenesis. Song et al, Proc, Nat Acad, Sci, USA, 84:6820-6824 (1987). A two-step process for homologous recombination in human cells is described. R ubnitz and 5ubra+iani (1984) Mo1. Ce1l.

Biol, 4:2253-2258, is essential for homologous recombination in mammalian cells. Kanamehonshuurio the next leopard The methods and compositions may be used to modify mammalian chromosomes, particularly to genomic target sequences. Using constructs with homologous sequences and at least one sequence homologous to the repeat sequence by providing for introducing deletions. Homologous recombination using chromosomal DNA deletions between the target sequence and the repetitive sequences within the chromosome by providing It is possible to obtain clones with the same DNA. Therefore, damage (lesi on) can be introduced into a YAC in yeast, and the YAC can be for homologous recombination and replacement of chromosomal DNA by modified YAC DNA of can be transformed into mammalian cells.

The theory of Toyosho Shohō■ How to use the composition of DNA &lllll Seiko and the cell composition Provide to qualify. This DNA composition contains sequences homologous to the target sequence, a sequence homologous to the same repeat sequence, and usually a bridge between the two sequences. It is a construction that This construct can be used to transform chromosomal DNA within the target cell host into other mutations. Although it is also possible to modify the use This modification can occur in two steps. In the first stage , a yeast artificial chromosome comprising a large NA fragment ial chromosome (“YAC”)) using the above DNA construct. modified by recombination. In the second step, two YACs or modified fragments are transform into the target host and scan the resulting cells for the presence of the desired lesion. Clean.

The subject methods described above may be used in eukaryotic host cells, particularly in mammalian hosts, and more particularly in primates. For use in a host, eg, a human. The above steps, to some extent, , although it can vary depending on the complexity of its genome, the nature of the repeats, etc. , the particular host is not critical to the invention.

As already indicated, the present DNA construct normally consists of at least two parts, more Normally, there are three parts: a homologous target DNA sequence; a homologous repeat sequence; and a bridge. will consist of. This homologous target DNA sequence can be any target gene, or to sequences adjacent to it, where the gene has been modified, particularly deleted. It can be what it should be. This target sequence can be a coding sequence or a non-coding sequence. May be intron, exon, 5 - or 3°-untranslated region. This homologous target sequence It lies close to the gene, usually within the target gene of about 250 kb or more. and upstream or downstream, usually upstream or downstream of the target gene. can be found in Therefore, this target region is typically approximately 250k b or more target genes and flanking sequences. The above construct The target sequence will normally comprise at least a portion of the target region; and can extend substantially beyond the target area.

The homologous target sequences described above will usually have at least about 70% homology, more usually will have at least about 80% homology, and more than 90% homology. It can also be a target. This homologous portion should be at least 15 nucleotides, such as will normally comprise at least about 30 nucleotides and will contain 0 ．． It can be greater than or equal to 5 kb, typically less than about 5 kb.

The homologous repeat sequences described above will usually be at least about 15 base pairs, more usually , at least 30 base pairs, and ti! i has less than about 1 base pair, more commonly will be less than about 0.5 base pairs. This homology is at least about 60% , more usually will be at least about 70%, and certain repetitive sequences 90% or higher. However, the opposite As the sequence changes, the sequence homology of the construct also varies within its target region. It will vary depending on the sequence of the repeat sequence. This construct is homologous to repetitive sequences. may comprise one or more repeat sequences, where the repeat sequences may be the same or different. It may be. Typically, this construct can be repeated or its Up to about 5 that can be homologous to different repeat sequences encountered within the host genome will contain repeated sequences of

The repetitive sequences in question are the Alu, LL, α-satellites found in humans. (a-5atellite), terminal pellets (telomertc) or subterminal pellets Subtelomeric repeats; and found in other mammals Contains similar sequences.

11 (bridge) above, if present, can serve various purposes. can. It serves to separate the target homologous sequences and repetitive homologous sequences mentioned above. be able to. It also allows for the incorporation of that sequence by selection for markers. One or more markers can be included to enable detection of the symptoms. Many kinds A variety of markers exist that can be used for various toxic agents, e.g. antibiotics, heavy metals, provide resistance to sequences, eg, excision sequences, primer sites, origins of replication, etc. The circular construct When used, markers include one or both of the above homologous sequences. You can stay on the bridge. When linear transverse structures are used for transformation, Normally this marker will be located between the homologous sequences described above.

The above constructs may contain an origin of replication for cloning, and viral functional sequences. transfection, integration, autonomous replication (autonomous) s replication (ars), separation (segregation) , and other chromosomal functions.

Constructs used for transformation usually have a base length of at least about 0.25 pairs, more usually will be at least about 0.5 base pairs, and 10 can be up to about 20 base pairs or more, typically up to about 20 base pairs. Ru.

A wide variety of target genes may be of interest depending on the purpose of the construct. Ru. For example, target genes include oncogene 3, immunoglobulin members of the immunoglobulin gene superfamily, especially the immunoglobulin gene family -constant and binding regions of -, surface membrane receptors, major histocompatibility complex antigens, can include microglobulins, enzymes such as kinases, lipases, etc. or they may be non-coding parts of genes, e.g. upstream regulatory elements .

The constructs will be prepared according to conventional methods to replicate in prokaryotic hosts. vectors capable of can be used. Normally, this vector is used to insert the various fragments in question. It would also include a polylinker for. This fragment can be used by any convenient means For example, restriction enzyme treatment from natural sources, synthesis, polymerase chain reaction (" PCR”) etc., and the end is inserted into the vector. If unsuitable for insertion, ligation with a suitable linker, blunt end ligation. It can be modified by etc. After each insertion, clone the resulting construct. sequence and analyze by restriction enzyme analysis, sequencing, or other convenient means. be able to. Once a construct is made, it can be cloned and expanded. produced and then modified as appropriate, often in its prokaryotic DNA. Remove the main part of A. Typically, this construct will be constructed whether it is an N-vector or If a 0-vector can be formed, it may be used in linear form. cormorant. Therefore, when hybridized, homologous sequences having a transverse sequence between the 3°-end of the sequence and the 5″-end of its downstream homologous sequence; or, alternatively, the 5'-end of its upstream homologous sequence and its downstream homologous sequence. can be linked to the 3'-terminal sequence of.

There are - or two transformation steps, depending on whether a one- or two-step approach is adopted. There will be. (“Transformation” refers to the transformation of exogenous cells into living cells while maintaining their viability). Refers to any means for introducing DNA. Therefore, transformation requires conjugation. transfection, transformation, transfection, e.g. calcium phosphate coprecipitation , cell fusion, protoplast or spheroplast fusion, DNA-coated particles Biolistics, lipofection, electroporation using offspring , microinjection, etc.). DNA is single-stranded or double-stranded It may be linear or circular. Transform the construct into the target host using conventional methods A two-step process is recommended when directly introducing the construct into the target host by That would be preferable.

In a two-step step, the target DNA is cloned into a YAC in a suitable yeast host.

This clone contains the target DNA, a particular group of clones or a library, as appropriate. It is known for that. Typically, this target DNA is at least about 50 kbp, and more Usually at least about 200 kbp, and usually less than about 1000 kbp. cormorant. Various yeast hosts, e.g. Pombi (Kage Tomomi L) etc. can be adopted. Conveniently, the host has access to at least one metabolic pathway. and auxotrophic for two or more metabolic pathways. good. At least one of these metabolic pathways is linked to genes present on YAC. The increased complementation ensures maintenance of the YAC within the host. the remaining money Metabolism dependence can be determined by insertion of a targeting vector into YAC or essential nutrients in the medium. can be supplemented by the supply of

- The origin of the DNA as DNA in the secondary cell or YAC is any subject mammalian cell, More specifically, it may be a primate cell, in particular a human cell, where a human cell may be an embryonic cell or a human cell. may be normal cells, including neoplastic cells, in particular normal cells. -as a secondary cell various cell types, e.g. fibroblasts, especially diploid dermal fibroblasts, keratinosa cells, myoblasts, lymphocytes, dahlia, epithelial cells, neurons, endothelial cells, etc. Alternatively, other somatic cells or germ cells may be employed. Especially targets the skin Fibroblasts, myoblasts, T-cells, retinal pigmented epithelial cells, etc. cells that can be easily proliferated and provide large amounts of normal cells, liver and kidney cells, etc. be. These cells may or may not express the gene of interest. target gene In situations where the target is inducible or expressed only in certain differentiated cells, Cells in which the gene is expressed can be selected, which means that they can grow in culture. May require dead cells.

YACs are prepared using conventional methods. Genomic DNA enzymatically, 8! mechanical or its cut by other means, usually at least about 50 kbp + usually less at least about 100 kbp, conveniently at least about 200 kbp, and usually about Fragments that are less than 2,000 kbp, more usually less than about 1,000 kbp Serve the food. This genomic DNA is inserted into a YAC and then the presence of this target gene is screen using an appropriate probe to identify the The existence of YAC is Y This can be confirmed by selective media for the markers present on the AC. YA Yeast cells containing C or YAC libraries were analyzed by hybridization analysis or can be characterized by polymerase chain reaction (PCR) using primers. Ru. The identified YACs are then used for recruitment.

YACs are usually transferred from their original yeast host to another yeast host convenient for manipulation. It will be. The new host has a variety of genes in it that allow selection by complementation. Single with multiple, usually at least 2, and possibly 5 or more mutations Or maybe a double stock. All yeast DNA from the original yeast host is transferred to the host for manipulation. The cells may be transformed into yeast cells or cells for sphnyloplasts that can serve as sphnyloplasts.

The resulting transformant was transformed to lack the complementary marker present on the YAC. cells may be plated on a selective medium to screen for cells.

On the other hand, YACs may be transferred by genetic crosses. The recipient host for manipulation is usually A single strain with a genetic defect that would be complemented by the original yeast strain may be one of the binomial hosts. If diploid, allow recipient cells to sporulate and The ascospores are mixed with the original yeast host on a suitable medium that allows mating. If-ploid , and the mating type is opposed to the original host strain, then the cells are subjected to direct mating. It's fine. Hybrid cultures are based on complementation between non-allelic auxotrophic markers. Then select on a selective medium in which only hybrid hybrids grow.

The hybrids are then allowed to sporulate and e.g. Randomization using the expression of the heterozygous recessive drug resistance marker Yudan1 for selection Either select the mucospores or separate the tetrads using a micromanipulator. Let go. This meiotic product is then determined by the presence of YAC markers in the recipient strain and Subject to genetic analysis for the presence of genetic markers. The existence of YAC is hybrid It can be confirmed by diization or PCR analysis.

Increasing the number of YAC copies per yeast cell to increase the efficiency of transfection into mammalian cells It may be desirable to YACs in a suitable host strain that allows multiplex amplification of YACs. Can be used together with. For example, Sm1th et al.'s PNAS (1990 ) 87: 8242-8246, YAC is constructed into amplifying YAC. be suitably manipulated to provide suitable markers for the introduction of the structure. stomach. Amplifiable YACs increase the efficiency of gene targeting and homologous recombination when amplified. It can also be useful.

The construct can be used for fusion with a cloning host or for introduction of exogenous DNA into a yeast host. into the yeast host, preferably by any other common means employed for can be introduced. Provides complementation for metabolic mutations in yeast when employing yeast hosts It may be desirable to include such markers in the construct. LEU, Hls , TRP, IJI? ASLYS, ADH, etc. are common mutations. This is it The presence of the construct integrated into the YAC can then be screened for. The presence of markers builds Should not interfere with the intended purpose of the body and should be tolerated by the target host It is.

If desired, the yeast marker can be modified to create a construct that results in the deletion of the yeast marker. It may be excised by using homologous recombination. Appropriate yeast marker The linear DNA was removed by restriction enzyme digestion followed by isolation of the linear DNA, followed by ligation. The yeast marker-free construct can be regenerated by subjecting the yeast marker to yeast.

Modified YACs can be prepared by any convenient means such as gel electrophoresis, gradient density or speed centrifugation. , can be purified by chromosome selection, etc.

The outcome of recombination of the construct and DNA depends on the distance from the target sequence to the repetitive sequence. It would be a superposition deletion. When direct recombination with the target host is employed, a variety of A large number of colonies will usually be obtained, each containing a deletion of cormorant. Cells are then cloned by limiting dilution and targeted for the size of the deletion. The area may be subjected to analysis. Then select clones with the desired size deletion However, in most cases the desired knockout will be achieved regardless of the size of the deletion. may be sufficient to exist.

Regarding YAC, YAC can be processed by any suitable method including fusion, electroporation, etc. can be transformed into target mammalian cells by any means. Breastfeeding that enables selection of integrants The presence of a homologous selection marker in the construct ensures that the homologous conformation resulting in the deletion is Then, mammalian cells that have integrated the construct can be selected. Lack of target The loss is usually at least about 1 kbp, more usually at least about 5 kbp; Must exceed 20 kbp, may be 50 kbp or more.

In the presence of the marker, transformed cells have approximately 0.0 GH with selective medium or dialyzed serum containing 1-0.5 μM meI-) Rexade grown in T-free medium and other markers are present, e.g. The medium may contain about 0.1 to Ing/ml of G418. Resistance Ronnie can be isolated and then analyzed for the presence of the construct.

The resulting cell composition is a mixture of superimposed deletions, and depending on the gene spacing, In addition to the target gene, other genes may also be deleted. Also, variable, By removing groups of constant or connecting regions, the genetic repertoire of immunoglobulin conformations is The tree can be modified. In particular, universal donor cells for transplantation therapy For the purpose of creating MHC class I and/or class II Si region deletions, Long deletions of specific target conformations can be created. Cell composition is extremely rare. can be separated by extraction and obtain cells with identically sized deletions. can be cloned for this purpose. Cells may be used as appropriate.

The examples below are offered by way of illustration and not by way of limitation.

experiment Yeast strains and growth. Satsukaromysis cerevisiae strain AB1380 has long arms. pY contains the human factor IX (F9) gene approximately 40 kb away from the terminal pellet. Contains a 650kb yeast artificial chromosome (HYA32G5) based on AC4 (Kada et al. Aa+, J. Human Genet, 46: 95-106 (1990)), HYA32G5 was crossed with YPH252 (Sikors Ki and Hieter (1989) Genetics 122:19-27) , the diploids sporulated, and the tetrads divided.

A his3Δ20〇 diploid isolate (designated YPH599) was identified and HIS3 Enabled Hector to operate it. This isolate containing a 650 kbp YAC is Gettaive, MATa ura3-521ys2-801 ade2401 It had trp 1Δ1his 3Δ200. Transformation is an intervening deletion 3 μg of Notl linear plasmid for and 6 μg of 5all for terminal deletion. Lithium acetate procedure using linear plasmids (Ito et al. (1983) J.

Bacteriol, 153.163-168). trait Transformants were plated on minimally supplemented SD plates lacking histidine (Rose et al., Method s in Yeast Genetics, Col1d Spring) Iarb or Laboratory Press, Co1d Spring Harbo r+ NY), the colonies were purified, and then tryptophan or urinary The ability of the plants to grow in the absence of water was tested.

Vector construction. The starting vector for construction of the interstitial deletion vector is pRs30. 3 (Sikorski and Weter (1989), supra). this vector contains the gene HI33 and a polylinker from Bluescript.

Bacterial neomycin phosphotransferase (Npt■) gene in mammalian cells 1.1 from PMC1neopolA, which contains all sequences necessary for gene expression. 2kb Sal I/Xho I fragment (Thos+as and Cape cchi (1987) Ce1l 51.503-512) as a plant end link. cloned into the unique AstI site in ρR5303 by . The resulting plasmid is called pH1sN303. pLl, Clos derived from l The 4.0 kb Sacl fragment of the tuned LI element (in the factor A 6kb full-length LINE (1 element) cloned from the insertion site is used as a polylinker. It was inserted into the Sac 1 site in both directions. The resulting plasmid was transformed into plLla and It is called plLlb.

The F9 sequence was inserted into a plasmid containing genomic DNA containing exons 7 and 8 of the F9 gene. constructed from the 5.4 kb EcoRl fragment of dePTM6 (Yosh i taka et al. (1985) Biochemistry 24.3736- 3750). 3, lk from position 1120 to position 3178 of this fragment To amplify the fragment b (within exo 77) by polymerase chain reaction. A synthetic oligonucleotide was constructed for this purpose. This is plLla and pI l-1 The plant end was ligated to the Xho I and C1a I sites of When linearized, the 3' end of exon 7 was exposed. obtained The intervening deletion vectors are called pF9L1a and pF9L1b. Terminal deletion vector -pBP110 and 111 contain the same Ll elements cloned in two directions.

Pulsed field gel electrophoresis (PFGF) and restriction analysis. for regular use gel DNA and high molecular weight DNA for PFGF were prepared by established procedures. (Davis et al. (1980) Meth, Enzymol, 65, 404-41 1; Schwartz and Cantor (1984) Ce1l 37.67 -75) Electrophoretic karyotyping using a counter-clamped homogeneous electric field (CHEF) device I investigated using The following probes were used for hybridization: p 1.2kb Sal I/Xho I neo derived from MclneoρoCA Fragment (7hosas and Capech i (1987) supra): pB 2kb EcoRI/BamHI 8153 fragment derived from M483 (Hi Contains the entire coding region of the S3 gene); 270 bp 3 isolated from BluS amHl fragment (Deiniger et al. (1981) J, Mo1. Biol, 151.17-33); XRp14/All5I/CEN4 (6 , 0) derived from the 1.6 kb Xho I CENa fragment (Hieter (1983) Ce1140.381-392); and pF9L1a-derived 600 pb BamHIF9 fragment. Randomize radioactive Rahelization probes Prepared by primer extension as described by Feinberg and Vogelstein (1999). 83) Anal, Biochem.

132, 6-13), and Scene-Screen Plus (Dupont NEN ) or hybridized to Zeta-Probe (Bio-Rad) nylon membrane. .

The target YAC, HYA32C5, present in yeast strain YPH599 is It contained a 650 kb fragment of human DNA derived from the Xq27SN region (Wa da et al., (1990) Am, J, I'1uaan Genet, 46 ;95-106). This YAC has been shown to be stable and A complete copy of the gene for flocculation factor TX (Fs), an unnamed DNA marker. DXS102 and a part of gene IX (Fs). That in YAC Homologous recombination between these homologous sequences and the pF9L1 series plasmids resulted in a series of deletions. It is predicted that this will cause His-colonies derived from transformation of YPH599 - to determine whether a deletion has occurred due to the presence of a marker on the YAC characterized for.

YPl (599 5.3 kb homolog (pF9DV) and pF9L1 series His-colonies were obtained when transformed with a P9 target hector containing . The average transformation efficiency of pP9DV is 175 colonies/1 dg of DNA. and two stromal deletion vectors yielded 25 and 15 colonies/μg, respectively. generated. A total of 7000 colonies harboring pF9DV from several transformations. - obtained, 750 colonies from pF9L1a and 180 colonies from pF9L1b. Colonies were obtained. Many of those colonies were isolated and They were tested for their ability to grow in the absence of liptophan. 147/1 60 or 92% of the colonies were both isolated suggesting that YAC was present in each. It was positive for the marker.

YACs modified by yeast transformed with pF9L1a and pF9L1b To determine whether the cells contain is impregnated in low-melt agarose, treated to release chromosome-sized DNA, and and fractionated by PFGE. YP) 1599 contains a YAC that is 650 kb. is. When this strain is denatured with pF9DV, the Ω-type recombination is 5.6k b Expected to produce long YACs.

The properties of YAC conferred by transformants obtained from pFsL1 plasmid were determined. Tested. A total of 147 Trp”, His”, (Ira” types) were tested. Of the transformants, 123 (84%) were located at a position different from that found in YPH599. It had a YAC that moved to 1. Better understand the structure of modified YAC I) Transfer the NA in the gel showing the chromosome karyotype to a nylon membrane and The filter was hybridized with a Nuo-specific probe. This detailed exam is 8 This was done on 8 clones. Of these, 9 (10%) remain unchanged. 14 (16%) were larger than 650kb, 55 (62%) were smaller; 4 (5%) have detectable YAC and 6 (7%) have two different Contains YAC.

The larger YAC has its homologous sequence (Ll, , F9 or plasmid sequence) The introduction of the circular part of the plasmid into the plasmid results from recombination 4.

Since most of the derivative YACs are small, they must have undergone deletions. those carries the terminal genetic markers, TRP 1 and URA 3, so its deletion interstitial, and this type was further analyzed.

The deleted YACs are of different sizes. Maximum size of deleted YAC The size is 630kb and the minimum size is 150kb.

In many cases, more than one member of each individual type of deleted YAC is obtained; This indicates that those molecules are the result of specific events other than random events. suggest

Deletions: If deletions are a useful genetic tool, they can be removed by homologous recombination. It is necessary that the The inventor first demonstrated that the HR event occurs at the P9 end. I checked to see if it was. The introduced plasmid is contained in the EcoR1 site of the neo gene. Mareta. Homologous recombination of F9 using pF9L1a and pF9L1b was 8.6 It is expected to produce a kb EcoRl band. In addition, EcoRI Digestion is expected to produce internal bands of 4.6 kb and 3.6 kb, respectively. be measured. DNA from a representative set of transformants was digested with EcoRI, Plot hybridization was then performed using neoprobe. Carrying 13/23 deletion The pF9L1a Kataga transformant and the 21/33 pF9L1b transformant have a A predicted hand of homologous recombination was generated.

The inventor also sought to establish whether the deletion was the result of an HR at the Ll end. desired. Unambiguous determination of this feature requires restriction enzyme maps around individual L1 elements. I need to know. No such complete YAC map is available. Fortunately, F9 Much of the gene itself has been sequenced, and the L1 element is located at the 5° end of the gene. has been identified. The restriction enzyme map around this region is known (Yosh itake etc. (1985), 9. If this element is targeted by recombination. 630kb (7) YAC capacity 11! Sarel, pF9L1b is used. In this case, several independent transformants containing J[, 630 kb (7) YAC We found that these target the L1 element at the 5' end of the F9 gene. The results were tested. Homologous recombination (HR) at this site is 7.5k It is expected to generate the Sph l band of b. Individuals of this kind tested All of the derived cell lines had such a band.

The L1 sequence present in the deletion plasmid and the target L1 at the 5′ end of the F1 gene are It has at least two differences in the restriction enzyme site. Ll in the introduced plasmid is It had an EcoR1 site that was not shared by the target. Its target is plasmid-carrying It had a Stu1 site that was not present in L1. Therefore, the crossover site is can be localized to their restriction enzyme site differences. Crossover is L1 arrangement If it occurs in the 5′-region (A), it is a 4.8 kb EcoRr fragment. was generated. If the crossover occurs in the 3-region (B) of the L1 sequence, then This resulted in an 8.4 kb EcoRl band. 3 types from 630kb class Analysis of the cell lines of A shows that two of them are the result of crossover with A; It was shown that one was the result of crossover at site B. 5 on target Crossover to the left of the tuI site generates a 6.2 kb fragment, And the 4.5kb hand is due to the crossover to the right side of Stu 1 site. There will be. The results showed that two members of the 630kb class were located on the left side of the Stu1 site. and one to the left of the Stu1 site. - Indicates that you will receive a The combination of those results indicates that YAC in Llb-7 , Stul and EcoRr multiplicity. We conclude that this is the result. These results showed that a single 20 kb deletion caused F9 and This is clearly shown to be the result of homologous recombination involving the L1 element. Therefore, At least most of the remaining deletions are due to similar events involving other L1 elements. It is predicted that this will happen.

Repetitive elements present in human DNA can also generate a series of integrated deletions. It can also be used to ascertain whether the 2) Proc, Natl, Acad, Sci, USA: 79: 78 04-7808). HA from YPH599 was quenched by many different restriction enzymes. and plot hybridized using the Alu repetitive element probe ( Blurs.

Deininger (1981) i). Individual restriction enzymes are different A pattern was generated. The patterns produced by the individual enzymes are It is a finger print. This fingerprint has been altered as a result of the deletion. representative YACs carrying deletions of different sizes to determine whether was digested with EcoRl and plotted with labeled Blur 8. Ibrigated. The complexity of the banding pattern increases with progressively larger deletions. and the pattern observed is due to the successive loss of Alu-containing restriction fragments. It was suitable for the loss.

f of YAC: If the deletion series is actually the name state series, then Construct a restriction enzyme map of YAC that should correspond to maps generated by conventional methods. It is possible to use them to accomplish. The inventor has discovered that the deleted YA The DNA of series C was digested with SalI and filtered on a pulsed field gel. The fragments were separated and hybridized with whole Hi-DNA. 6 A 50kb intact YAC is approximately 40kb and 230.150.100. 90 and bands corresponding to unresolved doublets are shown. 20kb defect close to F9 The loss resulted in the disappearance of the 150 kb band and its replacement by a 130 kb band. Ta. A 60 kb deletion resulted in a corresponding decrease in the size of this band. F9 placement Based on this type of analysis, which orders the fragments from the sequence, the YAC 5alI at 30,260,360.450 and 610 kb away from the ntolomere It was possible to infer that the area was included. This information is for HYA32GS Corresponds to the Sal 1 map.

Ll activation by HR: L1 essentials targeted by interstitial deletion hectors The element was determined. The inventors have demonstrated that individual elements are not targeted with equal frequency. Observed. This non-random distribution in the selection of Lis to target introduces 1, or it is based on the homology of the L1 element in the target to F9. It may be a function of distance from the anchor site. To distinguish between those possibilities, The present inventor has developed a terminal deletion hex containing the same element as the L1 element in the pF9L1 series. Vollraca f15. (1988) Proc, Natl, Acad, Sci, IJSA85: 6027-6031; Pava (1990) Dish R, 87: 1300-1304) was used. H Is-colonies were generated with ρBPIII at a frequency of 19 colonies/1 μg. Ta. 100 colonies from individual transformations were tested for phenotype. . For pBPllo and pBP111 derived transformants, 90% and It was found that 89% were His", Trp", and Ura-, respectively. confirmed that the targeted YAC was present. 3 from pBPllo 2 independent transformants and 24 independent transformants from pBPll. It was further characterized by pulsed field gel and Sasan analysis. 30/32 and 23/24 were found to contain recombinant products of several different sizes. (as demonstrated by probing with CEN 4). In the following table Data show the location and recovery of L1 elements targeted by different vectors. Summarize the number of times for each individual class. The size of the L1 element (sometimes depending on the vectors of both classes) some were targeted by one class of vectors, while others were targeted by only one class of vectors.

table Plasmid pBPllo pF9L1a pF9L2b pBP111*2 total The number is present in the direction from Trp to the Ura gene, where F9 exon 7 is 590 and the L1 sequence at 640. The number is exon 7 of the factor ■ gene. The kb of et al.

The above results demonstrate that homologous recombination occurs at one site with known target sequences and vectors for homology. – occurs by having a repetitive sequence at another site on the Nested groups of deletions of various sizes can be obtained based on site separation. Show that you can do it. In this embodiment, without knowing the entire sequence of the gene, Various genes can be elucidated. Produced by stromal and terminal deletion method Deletions have many uses. The deletion endpoint is adjacent to the vector sequence, so the site of the deletion It is possible to use unique DNA sequences at different positions.

Such unique sequences are used as boundaries in human genetic maps. It could act as a column tag site (STS). YAC to mammalian cells By introducing the gene transfer, the resulting gene transfer has the ability to act over long distances. allows the study of putative regulatory elements with force. Interstitial Grand Prize goes to YAC allows for the removal of sequences at certain positions, thereby allowing for genetic construct-functional analysis and Generates a support that can be used for long-term genetic interactions. the observation The result is that repetitive elements can be targeted by homologous sequences in either orientation. and Targeting of repetitive elements preferably involves At the same time, there is a targeting rationale for distinguishing between repetitive elements. You can get the frequency that suits you. The data consists of repetitive elements and targeted sequences. We show that homology and distance between the cells play a role in the generation of interstitial deletions. Therefore, In addition to providing elucidation of specific genes, the methodology of the present invention also provides It can also be used for

All publications and patent applications mentioned in this specification may be referred to as individual publications or patent applications. The same reference citation indicates that the application is specifically and individually incorporated by reference. This document is incorporated herein by reference.

Although the invention has been fully described, many changes and modifications may be made within the scope of the invention. It will be clear to those skilled in the art that this can be done.

Procedural amendment January 11, 1994

Claims (12)

[Claims] 1. A method of introducing mutations at targeted chromosomal sites within target host cells. So: D at a target site bound to a DNA region homologous to a repetitive sequence of chromosomal DNA. transforming various cells with a construct comprising a DHA region homologous to HA; This causes homologous recombination to occur within the target chromosome of some of the cells, and integrating the construct described above and causing the mutation of the chromosome described above, In one step, a YAC containing a yeast host with a target of at least 50 k base pairs. comprising chromosomal DNA is transformed with the above construct and the chromosomal DNA is such as can be done by bringing about mutations in the DNA, and hand, transforming the above target host cell with the above mutated chromosomal DNA, This allows the target chromosome to be mutated in some of the target host cells. and select for a target host cell comprising the above mutation. A method comprising: selecting. 2. 2. The method of claim 1, wherein said mutation is a deletion. 3. The mutations described above result in a series of superimposed deletions, and the deletions an additional step of separating at least one of the mutated cells from other mutated cells. 2. The method according to claim 1. 4. 2. The method of claim 1, wherein said construct comprises a selectable marker. 5. Claim 1, wherein said construct comprises a gene complementary to the yeast mutation. The method described in. 6. The target host cell is a human cell, and the repeat sequence is an Ll sequence, Alu sequence, α-satellite sequence, telomeric or sub 2. The method of claim 1, wherein the method is an array of terminal pellets. 7. According to claim 1, the target cell is a rodent cell. Method described. 8. 2. The target cell is an embryonic cell. the method of. 9. A method of introducing mutations at targeted chromosomal sites within target host cells. So; staining at target sites bound to DNA regions homologous to repetitive sequences of chromosomal DNA A construct comprising a DNA region homologous to chromoplast DNA allows comprising a YAC comprising a chromosomal DNA fragment of at least about 50 k base pairs transforming a yeast cell into which the above construct is incorporated; bringing about mutations of its fragments; A target host mammalian cell is transformed with the above mutated fragment containing the above construct. transmute; and selecting for a target host cell comprising the above mutation. Method. 10. 10. The method of claim 9, wherein said mutation is a deletion. 11. said target host cell is a human cell and said repeat sequence is an Ll sequence. , Alu sequence, α-satellite sequence, telomeric or sub-satellite sequence. 11. The method of claim 10, wherein the method is an array of terminal grains. 12. Regions homologous to the structural genes of the mammalian host chromosomes and the chromosomes of the mammalian host comprising regions homologous to the chromopolytic repeat sequences, and the two regions in the mammalian host. A DNA sequence that is joined by something other than the native sequence that binds it.