JPH08507696A - Method for limited deletion of DNA - Google Patents

Abstract

  (57) [Summary] Methods and DNA vectors are provided that allow for simplified means for deleting large amounts of mammalian chromosomal DNA. The vector comprises a positive selection marker, a negative selection marker, a region of homology for targeting, and a second region of homology for mediating the deletion.

Description

Detailed Description of the Invention                         Method for limited deletion of DNA                                   Introduction Technical field   The field of the invention relates to genomic modification of vertebrate genes by limited deletion of DNA. Itbackground   The ability to manipulate the vertebrate genome by homologous recombination allows the It became possible to change the gene structure. With the appropriate host cells, Using Nick Animal Technology to Permanently Change Germline DNA in Whole Animals it can.   The genome can be manipulated to add or delete DNA sequences. Chromosome DN Animals or cells with a targeted deletion of A have numerous uses. Relics that do not want to manifest Genes can be removed and the resulting cells can be used in therapeutic or experimental settings. I can.   By keeping one end point constant and changing the position of the second end point A series of deletions can be made in which the amount of DNA has been removed. Animals, especially humans When we are interested in determining the genetic map of It can be used for drawing.   Certain DNA sequences act as negative regulators of gene expression. Weak promoter Or enhancers and remove exogenous promoters near the transcription start site of the native gene. Use limited deletion of DNA to insert a strong promoter or enhancer Due to the pause of the gene Removal of certain upstream or downstream elements enhances expression of certain genes. May be sexualized. Or from the wild type gene placed upstream Strong promoters or enhancers can also be moved near the target gene .   In order to take full advantage of this technique, frequently and in some cases Vector D Genographs of limited size to create deletions without leaving behind the presence of NA sequences There is an interest in the ways in which a murine deletion can be created.Related literature   Scherer and Davis (1979)Proc. Natl. Acad. Sci. USA 76 (10): 4951-4955 Shows replacement of a chromosomal segment with an altered DNA sequence in yeast cells .   Mansour et al. (1988)Nature 336: 348-352 is a target for nonselectable genes. A general strategy for Ting (targeting) mutations is described. Thomas and Ca pecchi (1987)Cell 51: 503-512 and Thompson et al. (1989)Cell 56 (2): 313-321 Gene targeting to modify the HPRT gene in mouse embryo-derived stem cells Describes site-directed mutagenesis by.   Gene targeting methods for alterations in fetal stem cells are described in Valancius et al. And Smithies (1991)Mol. Cell Bio.11: 1402-1408 and Hasty et al. (1991)Natu re  350 (6315): 243-246.   Mombaerts and others (1991)Proc.Natl.Acad.Sci.USA 88 (8): 3084-3087 is the gene tag Size at the T cell antigen receptor β subunit locus by targeting The creation of a unique genomic deletion is described.                               Summary of the Invention   Simplified limited deletion or restriction of vertebrate chromosomal DNA using homologous recombination Methods and DNA constructs for constant mutations are provided. Substitution targeting vector Targets markers for positive and negative selection, to target specific sites And a region of additional homology to mediate the deletion.                            Brief description of the drawings   FIG. 1 shows targeting constructs and targets as described in the Examples below. It is an illustration of a chromosome.   FIG. 2 illustrates a bidirectional targeting construct, as described in the Examples below. And a diagram of the target chromosome.   FIG. 3 is a diagram of successive stages of deletion as described below.   FIG. 4 shows J and constants of immunoglobulin kappa light chains as described in the Examples below. Diagram of targeting constructs and targeting experiments for constant region inactivation is there.   FIG. 5 shows J and constants of immunoglobulin kappa light chains as described in the Examples below. It is an illustration of preparation of a vector for inactivating a constant region.   FIG. 6 shows J and constants of immunoglobulin kappa light chains as described in the Examples below. 1 is a diagram of the final deletion vector for inactivating the constant region.   FIG. 7 shows the J and constant region Southern components of the light chain as described in the Examples below. It is an illustration of analysis.                          Description of specific embodiments   DNA constructs for homologous targeting by substitution of DNA The method used introduces localized lesions at specific sites in the vertebrate chromosome. By "damage" is meant a change in the DNA sequence at that site. Targeting Use the construct to leave the vector DNA sequence in the targeted chromosome. It is possible to delete a segment of chromosomal DNA. The method is a vector Also used to alter a sequence in a target chromosome without leaving the DNA sequence behind Can be.   Targeting constructs include positive selectable markers, negative selectable markers (these are May be the same marker in some cases), homology to the target site in the chromosome With a sequence from the region outside the sequence to be targeted in the chromosome Consists of additional homologous sequences. Therefore, the target site in the chromosome must be homologous in the vector. Limited by row. Additional homologous DNA sequences (ADH; additional DNA homology) A sequence that is on the same chromosome as the target sequence, and this additional sequence is upstream on the chromosome. It can be located either downstream or downstream. Finally the ADH sequence on the chromosome The DNA sequence between and the target sequence will be deleted.   Preferred targeting constructs for use in the present invention are "omega" or "substitution". It is a targeting vector. However, it provides a homologous sequence for recombination An "O" or "insert" target if a single target sequence is sufficient for You may use a vector.   The targeting construct is introduced into the host cell, preferably after linearization, Here it undergoes homologous recombination and is integrated at the site of the target sequence. Each element in the linear composition The disposition of the elements is shown in FIG. However, an exemplary order of functional units is shown And the order of functional sequences between target sequences may vary depending on the particular intended use. Let's place Good. Moreover, target by homologous sequences in the targeting construct. The intended chromosomal sequences may be contiguous or separated. If separated , Homologous recombination by double cross-over will result in the initial deletion.   For convenience, the DNA on the targeting construct that is homologous to the DNA sequence in the chromosome Elements are called vectors or “v” homologous sequences and chromosomal counterparts are chromosomal sequences or Will be referred to as "c". The two v-target sequences required for homologous recombination are homologous. At the extreme 5'and 3'ends of the normal targeting construct that form the arm of the there will be. The exception is when using positive-negative selection at the homologous recombination stage. Yes, for example, the negative selectable marker can be the terminal sequence. "C-ADH" or "V-ADH" refers to the phase in a chromosome or vector used to achieve a limited deletion. Say the same array.   The double crossing-over phenomenon at the target site in the chromosome is an issue as long as the c-target sequence is not continuous. Of the chromosomal region between the two c-target sequences and the two v-target homology arms. Results in the replacement of the intervening constituent sequence. Sequences that are not functional in the vertebrate host, For example, the bacterial origin of replication and antibiotic resistance sequences may be outside or inside the construct arm. Can be on the side. Sequences located outside the arm are integrated into the chromosome And the sequence placed between the arms will be integrated into the chromosome. U Selectable markers for the v-ADH region and vertebrate cells are two homology arms Will be placed between   After introducing the constituent DNA into the target cells, the selection pressure is adjusted by the positive selection marker. It is possible to grow only those cells that contain It is possible. The chromosome in which the construct is integrated is the "targeted chromosome". As shown in FIG. Diagram of "chromosomes" shows the c-target sequence as a continuum But above It should be understood that they may be segregated on the chromosome as described above. Embedded After that, there will be one copy of each target sequence and two copies of the ADH sequence. IADH The pea is chromosomal in origin (c-ADH) and the other ADH sequence is the target (V-ADH). The two copies should be at least v-ADH And a negative selectable marker for the targeting construct between the sequence and the c-ADH sequence. They are separated by a color body array.   If the cells are positively selected, the addition of negative selection medium to the cells, for example, When car is a herpes thymidine kinase gene, using ganciclovir, Cells can be immediately negatively selected. Under negative selection conditions, between the target sequences Cells carrying the placed negative selectable marker will usually die. Homology c-AD Loss of negative selectable marker due to intramolecular crossover involving H and v-ADH sequences. Cells can grow under negative selection conditions. Or positively selected Screen cells to identify clones targeted by homologous recombination. Targeted clones can also be subjected to negative selection. Depends on the absolute efficiency of the deletion resulting from the first intermolecular homologous recombination step and intramolecular crossover. Therefore, a second independent negative selection step may be necessary. As stated, the The method involves v-targeting to increase the efficiency of the intermolecular homology targeting step. Do not use negative selection with a negative selection marker different from the one placed between the rows. Can also be.   Since the c-ADH and v-ADH sequences are on the same nucleic acid molecule, the deletion is a molecular It is an internal recombination phenomenon and will have a higher efficiency than homologous recombination which is intermolecular. Therefore, the deletion phenomenon expected in the present invention is directly affected by intermolecular homologous recombination. Is much more likely to provide a large DNA deletion.   An example of the order of elements in the composition is depicted in FIG. 5'end and 3'end It will usually be bordered by the v-target sequence. v-ADH sequence and selectable marker It will be located between the v-target sequences. v-target sequence and v-ADH sequence are all the same Oriented, that is, the orientation of the DNA of the individual components in the construct is such that it is on the chromosome The same as at some times, either 5 '→ 3' or 3 '→ 5'.   Negative selectable markers oriented in either transcriptional direction are v-ADH on the construct. It will be located between the sequence and one of the v-target sequences. The exact position is c-ADH It will depend on the position of the row. On targeted chromosomes, negative selection markers The car is located between c-ADH and v-ADH.   The location of the positive selectable marker on the construct will depend on the desired end product. Because the excision stage leaves behind or leaves a positive marker in the chromosomal locus. There are times when you don't. If both positive and negative selectable markers are targeted Negative if placed between c-ADH and v-ADH in the stained chromosome Both will be deleted when drug selection is applied. The negative selection marker is c-ADH And v-ADH and the positive selectable marker is on the outside, the excision step is It will leave a positive selectable marker in the chromosome of the deletion locus. In this situation, It may be preferable to apply both positive and negative selection during the elimination step. General In addition, the positive selectable marker may be oriented in either transcription direction. Positive selection Similar to markers, the targeting of c-ADH and v-ADH in chromosomes Any exogenous sequence from the non-interstitial construct will be in that position after the event of final recombination. Will remain.   The arrangement of elements on the chromosome would be as shown in FIG. The two c-target sequences are , Next to each other to facilitate the first double crossing phenomenon It may be contiguous or separated for deletion by homologous recombination. Hope Preferably, at most 20 kb between target sequences on the native chromosome, usually at most about 10 kb, Preferably there will be at most about 1 kb of nucleotide distance. C-ADH on the chromosome The distance between the c-target sequences will depend on the desired end product. Usually this distance Would be much larger than the distance between the two c-target sequences. It is 4000 kb long Can be any number, usually at most about 100 kb in length, and usually at least Would also be about 500 bp in length.   The length of the homologous v-target sequence and the v-ADH sequence should be at least about 50 nucleotides in length. Preferably at least about 100 nucleotides in length, usually at least about 0.5 nucleotides in length. kb and a length of less than about 100 kb, usually less than about 20 kb, preferably less than about 1 kh. Let's do it. The homology region should be at least about 90%, preferably less than the sequence of the native chromosome. It will have at least about 95%, and more preferably at least about 99% sequence identity.   The v-target sequence may be a subtle change in the region of homology, such as the introduction of a restriction site, the Sequence of the gene locus or promoter locus, introduction of splice site or Can be prepared for removal and the like. Subtle changes are the base pairs involved in the target sequence. Substitutions less than 10 bp total such that the percentage does not exceed 10%, usually 5%. And / or delete.   Various markers are available for selection. HPRT Mini Remains Gene [Reid et al. (1990)Proc. Natl. Acad. Sci.87: 4299-4303], against G418 For resistanceneoHSV Timiji for susceptibility to the gene ganciclovir And a hygromycin resistance gene. Positive selection and negative Markers that can be used for both selections may be used, eg HPRT. In this case, - The car must be placed in the construct as a negative selectable marker. Or , For positive selection egneo, Hygromycin resistance, etc., and for negative selection eg HSV -Separate markers such as TK, cytosine deaminase, etc. may be used.   Another element of the construct is the polymerase chain reaction (PC) for identifying recombinants. R) sequences encoding specific primer regions that can be used in R), gel electrophoresis Addition of one or more restriction sites taking into account identification by To remove target restriction sites or identify target cells that have undergone the desired modification. Other changes can be included.   Various techniques can be used to introduce linear DNA into target cells. Wear. Such techniques include electroporation, calcium precipitation DN A, fusion, transfection, lipofection and the like. DNA The particular method of introduction into the cell is not critical to the invention, but the electroporation Preferred.   Once the target cells have been transformed, the cells are then plated in selective medium and selected. Selection with marker genes by growth in culture or by Southern analysis. You can Prepare for different size fragments depending on whether homologous recombination has occurred PCR can be used to analyze cells by using primers that will Can also be. In this way, target cells that have undergone the desired alterations can be identified.   Altered cells can also be identified by alterations in gene expression if appropriate . Increased gene if the deletion removes a negative regulatory element or silencer Transcription and protein synthesis would be expected. Alternatively, a gene or positive regulatory element If a component, such as an enhancer, is deleted, it causes decreased transcription and protein synthesis. Trying to see Would. The presence or absence of a gene product can be detected by using specific antibodies. Detect the presence or absence of mRNA from a gene by functional analysis of the product Therefore, it can be detected by, for example. The gene product is a surface membrane protein If desired, work with FACS in order to identify cells with the desired phenotype. Null antibodies can be used.   The subject methodology includes detailed structural genetic analysis of mammals, investigation of damage associated with genetic diseases, and And can be used to manipulate gene expression. Can be targeted Genes include β-globin, erythrocyte metabolic enzyme, complement system, coagulation factor, dys Of loffins, carbohydrates, lipids, amino acids, steroids, purines and pyrimidines Metabolic enzymes, transport proteins such as cystic fibrosis transmembrane regulators, immunoglobulins Genes, T cell receptor genes, histocompatibility antigens (major and minor), etc. You can   The subject method has advantages in certain situations where other techniques may not be sufficient. Can be found. Since the second step of the method is intramolecular, other methods are It is possible to achieve a deletion phenomenon that could not be achieved by the same recombination. Thus, for example, host organisms that produce endogenous immunoglobulin heavy and / or light chains. In order to eliminate the ability of the A combination of exons, such as heavy and / or light chain (κ and λ) loci, You can change the gene by inactivating the locus you want to inactivate. You can   Another application expresses surface major histocompatibility complex (MHC) antigens for transplantation The use of the method of the invention for the production of non-universal "donor cells". For example, The method can be used to delete some or substantially all of the MHC. Kura Class I and Class II MHC Antigens are heterodimers, each consisting of an α subunit and a β subunit. Ku In the Ras I MHC antigen, the β subunit is β₂-Microglobulin. Special The focus is on subunits of MHC antigens (eg β₂-Microglobulin Of at least one copy, preferably both copies. Alternatively, use this method to delete another gene that affects the expression of MHC antigens It can be performed. For example, a gene that regulates MHC antigen expression, such as MHC TAP1, TAP2, LMP2, and LMP7 residues in class II loci that regulate antigen-dependent presentation The gene can be deleted to prevent MHC presentation on mammalian cells.   Depending on the nature of the mammalian cells, the number of competent MHC antigens may be low. Cells that lack at least one can be used as a donor to an allogeneic host. Or, if they are fetal stem cells, use themselves as a source of organs for transplantation It can be used to create a chimeric mammalian host capable of Pay particular attention Of at least one MHC of class I or class II, preferably class I This is a method for preparing cells lacking an antigen. The cells may vary in viable hosts. Can perform various functions. This method is₂-For the microglobulin gene One of the contiguous loci, the α-subunit of class I or II MHC antigens. To regulate the expression of β-subunit of class II MHC antigen, or MHC antigen Targeting constructs of the invention for deleting DNA in related genes Transfection of mammalian cells of a predetermined species (especially normal cells) by including. The targeting construct may prevent expression of a functional MHC antigen molecule, Deletion that causes the deletion in at least one copy of the native gene, usually both. Will create loss. Deletions made in only one copy of the gene to be inactivated. When Cells with a single, unmutated copy of the genetic gene, and then to a second transformation You can call. In this case, the second deletion may be the same as the first damage but different. You may. Screening the resulting transformants for the absence of functional target antigen And further screen the cell's DNA for the absence of the wild-type target gene. Can be confirmed. Alternatively, by mating hosts that are heterozygous for the mutation By doing so, it is possible to obtain homozygosity for the phenotype.   The cells that can be transformed are any mammalian cells of interest Often, these are used for cell therapy, investigations, interactions with other cells in vitro, etc. Can be Among the cells of particular interest are, among other lineages, Langerha Islets, adrenal medulla cells that can secrete dopamine, osteoblasts, osteoclasts , Epithelial cells, endothelial cells, T lymphocytes, nerve cells, glial cells, ganglion cells, retina Cells, fetal stem cells, hepatocytes, bone marrow cells and myoblast (muscle) cells. So These cells can be used in mice and other rodents, rabbits, pigs, cats, cattle, dogs, humans, etc. Can be obtained from any mammalian cell containing.   Cells from patients with defective lymphocytic syndrome are treated by conventional methods such as sorting, affinity. Column, magnetic beads and the like. CD 3, 4, 8, 10, 15 also Uses a monoclonal antibody against a marker such as 19 to detect lymphoid cell type B By using a monoclonal antibody specific for cells or T cells, the desired It is possible to isolate cell populations and their progenitor cells in a substantially homogeneous composition. Wear. Similar to MHC antigen-deficient cells produced by homologous recombination Transmission deficient cells can be utilized.   MHC antigen-deficient cells have been selected for specific functions and have been found in mammalian hosts. Introduced or used for research or other purposes Will be. Stem cells that act as progenitors of any of the above cells will also be of interest U This is a progenitor cell that is already directed to a particular lineage There may be. Of particular interest are epidermal cells such as keratinocytes and retinal epithelial cells. , Myoblasts, hematopoietic cells, and can be easily manipulated in vitro, in culture Can be maintained for a long period of time and introduced into a host where the cells May be other cells that are viable and functional for long periods of time.   For fetal stem cells, fetal stem cell lines can be used, or Mussels, such as mice, rats, guinea pigs, Chinese hamsters or Fetal stem cells can be newly obtained from another small experimental animal. Those cells Proliferated or leukemia inhibitory factor (LIF) on a suitable fibroblast feeder layer Can be grown in the presence of and then used for mutation.   The targeting constructs of the invention can be used to prepare, amplify, transform host cells. Functions available in transformation and integration of the construct into host cells Can be modified to include specific entities. The available technology is phosphoric acid Lucium / DNA co-precipitate, microinjection of DNA into the nucleus, electron Cloning, bacterial protoplast fusion with whole cells, transfection And so on. DNA can be single-stranded or double-stranded, linear or circular, relaxed or super It can be coiled DNA. A variety of methods for transforming mammalian cells For technology, Keown et al.Methods in Enzymology(1990) 185: 527-537. That. The construct has been prepared and manipulated, and unwanted sequences from the vector, such as Once the unwanted bacterial sequences have been removed, the DNA construct can be immediately transferred to target cells. Can be introduced. As already pointed out, the DNA is introduced into target cells Any convenient technique for can be utilized. Standard After transformation of the target cells, the positive and / or negative marker Multiple targets using Omicin resistance and Acyclovir or Ganciclovir resistance The cells are selected. Those cells exhibiting the desired phenotype are then subjected to restriction analysis, electrical Further analysis is performed by electrophoresis, Southern analysis, polymerase chain reaction and the like. Then generate The transformed cells are selected by the absence of the target MHC antigen on the surface of the cells. This This can be achieved in various ways. For example, with complement, of the target MHC antigen Antibodies to either epitope can be used to kill cells bearing the antigen it can. Alternatively, a suitable antibody, especially a monoclonal antibody, and a toxin such as A conjugate with a chain A, abrin, diphtheria toxin, etc. can be used. Affi Nity chromatography may be used, in this case comprising the target antigen Antibodies can be used to remove cells. The cells that survive as a result Introduced in vivo lacking at least one MHC antigen on their surface Occasionally, it will not undergo transplant rejection as much as wild-type cells.   Then, the cells are grown in an appropriate growth nutrient medium and used in various forms. Can be. The cells can be used for transplantation to become part of, or augmented with, existing tissue. It can be propagated to produce tissue for transplantation into a heterologous host. For example, keratinocytes Can be used to replace skin in case of burns, in which case keratin prior to application The cells are grown to form a continuous layer. Similarly, keratinocytes may be used elsewhere. Used in plastic surgery to replace skin removed from the host in order to You can Other uses for keratinocytes include transplantation in the case of pressure ulcers. It   In the case of the islets of Langerhans, they are propagated and for insulin production In a capsule or other state for insertion into the host Can be introduced at. In the case of retinal epithelial cells, it cures visual disorders such as macular degeneration. They can be injected into the subretinal space of the eye for treatment. In the case of immune cells, They can be injected into the bloodstream or into something to treat immunodeficiency. Myoblast In the case of cells, to treat muscle wasting diseases such as Duchenne muscular dystrophy , They can be injected at various sites. Due to organ transplantation, between closely related species Can use non-syngeneic tissues such as heart or liver xenografts.   Depending on the nature of the cell, the treatment applied and the disease, the cell will be used as a thin film. Can be introduced into the container for maintenance at a specific site, Or as a solid material or matrix impregnated in an inert matrix Can be used independently. The number of cells administered will depend on the particular application and Vesicles will vary widely depending on the method of administration. Administration is in the right place Can be by injection, topical application, ablation and placement.   Another situation was the introduction of a sequence at the target site that is resistant to homologous recombination. That is the case. By using the subject methodology, the The sequence can be introduced into the desired location and contains a resistance locus. However, the region between the insertion site and the resistance locus can be deleted by using the ADH sequence. You can Thus, for example, in order to activate the expression of the gene of interest, An enhancer, a promoter, an exon, etc. can be introduced into the position. Therefore , The subject methodology is that one locus is resistant to homologous recombination and Deleting the region close to the resistance locus while allowing the sex sequence to approach the resistance locus Close to the resistance locus that would be affected by the sequence of interest if desired It can be used to introduce a sequence of interest into a site.   The target cells may be various vertebrate cells, especially animal cells, more preferably mammalian cells. Either may be used. Of particular interest is the production of transgenic animals. Can be used to introduce an altered chromosome into the germ line of the host animal Fetal stem cells that can be used to Fetal cells of particular interest Include rodent cells such as mouse, rat and guinea pig cells .   The method of the invention can be used to produce deletions in chromosomes of various sizes. Wear. Relatively small deletions (500 bp to 15 kb) can be achieved in a limited fashion. Such small deletions select the target region for recombination that is separated by the desired deletion. Can also be achieved by standard targeting methods. However, using standard methodology, the presence of a positive marker in the final locus is It must be tolerated. When you don't want any extra markers at the final locus, The method of the invention will allow the deletion to be made without leaving exogenous sequences behind. Let's do it.   Large deletions up to 4000 kb can be achieved by the methodology of the invention. The size of the deletions that can be made by standard methodologies are limited and differ in efficiency. Become. The method of the present invention is much more powerful than the prior art. Because the present invention is Chromosomes using negative selection rather than relying on intermolecular homologous recombination for the excision step This is because they rely on internal recombination. The large deletion phenomenon resulting from the present invention is the standard target Compared to the deletion phenomenon resulting from the Ting experiment, at a frequency within the range of a typical experiment. It is likely to happen.   In some cases, deletions of long chains of DNA may result in deletion of the targeting construct. It may delete genes that are essential for cell viability. In such cases, Second transfection via targeting construct or with another vector Recessive lethality Essential genes, which can be genes, can be added in their original place in the chromosome. Lack Essential genes must be added in place before the body can be homozygous.   The method of the present invention can have numerous embodiments. 2 shows that An embodiment of the invention wherein a second negative selectable marker and an ADH sequence are included in the construct Is. The second v-ADH usually has no sequence identity with the first v-ADH right. The second c-ADH site has the same staining as the first c-ADH and c-target sequences. Located on the chromophore, the c-target sequence will be located between the two c-ADH sequences. After the initial targeting, negative selection is applied. This is the first c-ADH Will select for cells lacking the sequence between V and ADH. Hope A second round of negative selection performed for different markers was performed with the second v-ADH. One would select for cells lacking the sequence between c-ADH. Thus , The deletion will be bidirectional from the first targeting site.   Alternatively, a series of v-ADH sequences and a negative selectable marker sequence are targeted in a permuted format. Placed on the targeting construct and proceed from the first targeting site on the chromosome. Chromosomal DNA sequences can be deleted in successive steps in one direction ( (Figure 3). Thus, using the method of the invention, larger and larger contiguous DNA segments Can be deleted. Targets using the arrangement of elements as described herein. To create a bidirectional deletion and a gradual sequential unidirectional deletion, It can result in specific deletions in the chromosome.   In another embodiment, the ADH sequences can be repetitive sequences. In that case, the endpoint of the deletion would be at many different sites where repeat elements have been found so far. There can be. Thus, the starting point for a given deletion and the A series of reduction formulas with variable amounts of DNA Loss can be made. As the repetitive sequence of interest, Alu found in humans, L1, α-satellite, telomeric or subtelomeric repeats, and other mammals There are similar repetitive sequences found in the class.   The ADH sequence may be homologous to one of the target sequences, but the genetic damage or Includes mutations. Genetic damage or mutations are insertions, substitutions or deletions in a DNA sequence. Can be The number of modified nucleotides is usually less than about 20 bp and more Commonly less than about 10 bp and will be at least 1 bp. In this case, normal AD There is no chromosomal sequence between the H sequence and the target sequence, so the deleted sequence is a vector. -Only array. This way, you don't have to leave behind markers or vector sequences. Introductory mutations can be introduced.   The following examples are provided by way of illustration and not limitation.                                 Example     Inactivation of mouse immunoglobulin kappa light chain J and constant region A. Targeting experiment design   First delete the constant region and the J region of the kappa locus and flank the constant region A device for replacing it with three elements by homologous recombination using a homology region. A targeting vector was designed as a recombinant vector (Fig. 4). Some In the case of the diphtheria toxin gene (A Strand) was included as a negative selection marker. The three elements are the G418 resistance drug marker, Mouse DNA ADH sequence homologous to a region of the kappa locus upstream of the J region , And the thymidine kinase gene. Of the inclusion of ADH sequences in the vector As a result, this first targeting is a second copy of the ADH sequence in the locus. -I put it down. Use this copy Achieved selective deletion of sequence between two segments by applying selective pressure . In this case, the cells will have two segments to survive the ganciclovir selection. Delete the intervening thymidine kinase gene.B. Creation of targeting vector   129 Phases from mouse fetal liver genomic library (Stratagene, San Diego, CA) Homogeneous regions were derived and screened using two probes. First plow Is a 1.6 kb HpaI / BamHI fragment extending into the constant region [Steinmetz and Zachau (1980)N ucleic Acids Research 8: 1693-1706]. Hybridizes to this probe To identify isolated lambda phage clones and to purify and isolate phage DNA. used. Analysis of this DNA revealed that the HpaI / BamHI probe was high in the 5.6kb SphI / BamHI fragment. It was shown to have been bridized, and this fragment was then cloned into the SphI site of plasmid pUC218 and Ba Subcloning between the mHI sites gave the plasmid pUC218 / 5.6K. This subque The loan consists of the J region of the kappa light chain locus, the intron enhancer element and the constant Area included. The second probe is a 0.8 kb EcoRI fragment 2.8 kb upstream of the J region [Van  Ness et al. (1981),Cel27: 593-602]. Positive for this probe In the phage DNA from the λ clone, the probe hybridized to the 5.5 kb SacI fragment. It was shown to be soybean. Then, the fragment was digested with Sac of pBIuescript SKI (Stratagene). Subcloning into the I site gave the plasmid pSK.5'K (Fig. 5).   A 5'homology region flanked by diphtheria toxin genes in some cases, Thymidine kinase gene, ADH, neomycin resistance gene and 3'homology region The inactivation vector containing the region (Fig. 6) was transformed into three plasmids containing the following elements ( Figure 5): (a) mouse phosphoglyceride as a negative selectable marker. Tokinase gene Contains a diphtheria toxin gene (DT) driven by the (PGK) promoter Or 5'-homologous fragment not containing, (b) pMClNeo [Thomas and Capecchi (1987)Cell  51: 503-12] and the G418-selectable neomycin (neo) gene and AD Mouse phosphoglycerate kinase gene as a negative selection marker with H (PGK) promoter driven herpes thymidine kinase gene (t k), and (c) 3'homology breaks with or without PGK-operated DT gene One piece. All three of these plasmids (Fig. 4) were modified by changing the polylinker. From pSK.A, pSK.B and pSK.C derived from the plasmid pBluescript SKI Each was made. Insert the polylinker of plasmid pBluescript SKI into KpnI site and SacI Between parts Was modified by cloning the plasmid pSK.A, Modified by cloning a synthetic polylinker To generate plasmid pSK.C.   Diphtheria toxin gene is PGK promoter and bovine growth hormone polyadenylate Signal [Woychik et al. (1984),Proc. Natl. Acad．Sci. USA, 81: 3944-3948 Pfarr et al. (1986),DNA 5: 115-122] flanked by diphtheria toxin genes A set was made. Operated by the human metallothionein (hMTII) promoter Diphtheria PTH-1 containing a toxin A chain [Maxwell et al. (1986),Cancer Res.46: 4660-4664] These 2.3 kb XbaI / EcoRI fragments were cloned into pBluescript SKI cut with XbaI and EcoRI. By rolling, the plasmid pSK.DT was obtained. The hMTII promoter of pSK.DT was replaced with pKJ1 [T ybulewicz et al. (1991),Cell 65: 1153-1163]. Changed. Use the 0.5 kb XbaI / PstI fragment from pKJ1 as an oligonucleotide Use the PstI / NcoI adapter to ligate to the 3.1 kb XbaI / NcoI fragment from pSK.DT. , Plasmid pSK.pgkDT. Oligonucleo 248 bp containing the bovine growth hormone polyadenylation signal, obtained by breadth The fragment was cloned into pCR1000 (Invitron Corp., San Diego, CA). Next Then, this polyadenylation sequence was inserted into HindIII and HpaI digested pSK.pgkDT. Cloned behind the DT gene as a III / HpaI fragment and plasmid pSK.pgKDT I got bovGH. The DT gene cassette from pSK.pgkDTbovGH was oligonucleotied. Do 2.1 kb E in pSK.A cleaved with EcoRI and NotI using HindIII / NotI adapter Transferred as a coRI / HindIII fragment to obtain plasmid pSK.A / DT. pSK.A and pSK.A / DT The 5'homology region of the κ locus was clonined between both SphI and Bsu365 sites. I'm sorry For this, a partial Bsu36I digestion of the plasmid subclone pUC218 / 5.6K Then, the 4.0 kb SphI / Bsu36I fragment obtained from the complete SphI digestion was added to pSK.A or pS. K.A / DT was ligated to obtain plasmids pSK.A / 5'K and pSK.A / DT / 5'K, respectively. The In rasmid pSK.A / DT / 5'K, the 5'end of the DT gene and the 5'end of the K fragment are opposite. In the transcription direction of Close to.   Plasmid pKJtk [Tybulewicz et al. (1991),Cell65: 1153-1163] from PGKtk The gene is a 2.7 kb EcoRI / HindIII fragment with unique EcoRI site and HindIII region of pSK.B. Cloning between positions gave pSK.B / TK. The 0.8 kb EcoRI fragment used for ADH is pSK Cloned from .5'K, the tk gene and the 5'end of the K fragment are in opposite transcription directions. Plasmid pSK.B by ligating it into the EcoRI site of pSK.B / TK so that they are close to each other. /(TK/0.8K) was given. 1.1kb neo gene from pMClNeo as XhoI / BamHI fragment Clone between the same sites of pSK.B / (TK / 0.8K) to give pSK.B / (TK / 0.8K / Neo) It was PSK.C digested with BamHI and treated with alkaline phosphatase was added to pUC2 3'by ligation to the 1.1 kb BgIII / BamHI fragment isolated from 18 / 5.6K A plasmid pSK.C / 3'K containing a homologous fragment was created. In pSK.C / 3'K, κ disconnection Transcription begins in the plasmid polylinker from the SacI site to the KpnI site. Was oriented so. EcoRI / HindIII fragment of 2.1 kb DT cassette from pSK.pgkDTbovGH Was cloned into the same site of pSK.C to prepare pSK.C / 3'K / DT.   Three-part ligation was performed to create the final targeting plasmid (Figure 6). pSK. 4.0 kb NotI / NdeI fragment from A / 5'K, 4.8 kb NdeI / SacI fragment from pSK.B / (TK / 0.8K / Neo) A piece (obtained by partial SacI digestion of the above plasmid followed by NdeI digestion), and pS The 4.0 kb SacI / NotI fragment was isolated from K.C / 3'K and ligated together to create pK. (TK / 0 .8K / Neo) was created. 6.1 kb NotI / NdeI fragment from pSK.A / DT / 5'K, pSK.B / (TK / 0.8 K / Neo) to 4.8 kb NdeI / SacI fragment and pSK.C / 3'K to 4.0 kb SacI / NotI fragment. It was isolated and ligated together to make pK.DT / (TK / 0.8K / Neo). pSK.A / D 6.1kb NotI / NdeI fragment from T / 5'K, 4.8kb NdeI / SacI fragment from pSK.B / (TK / 0.8K / Neo) One piece, and And pSK.C / 3'K / DT to a 6.1 kb SacI / NotI fragment (SacI partial digestion of the plasmid followed by N (obtained by otI digestion) and ligated together to create pK.DT / (TK / 0.8K / Neo) / DT was created. Purified plasmid for electroporation The DNA is first digested with PvuI or ApaLI and then extracted with phenol / chloroform. It was taken out and precipitated by addition of ethanol, followed by centrifugation. Got Resuspend the DNA pellet in 10 mM Tris-HCl, 1 mM EDTA (TE) at a concentration of 1 mg / ml. It became cloudy.C. Introduction of DNA into cells   Fetal stem cell line E14-1, a subclone of E14 [Hooper et al., (1987)Nature 3 26: 292-295], 15% heat-inactivated fetal bovine serum, recombinant murine leukemia inhibitory factor ( Gibco BRL, ESGRO from MD, 1000 units / ml), β-mercaptoethanol (0.1 m M), glutamine (2 mM) and penicillin (100 U / ml) / streptomycin DMEM (JRH Biosciences, containing 4.5 g / l glucose supplemented with (0.1 mg / ml) Irvine, CA), 5% CO₂Grow at 37 ° C in. Essentially Koller And mitomycin treated as described by Smithies (1989) supra. The cells were cultured on a feeder layer of primary fetal fibroblasts. Fetal fibroblast Is β₂-Prepared from day 14 fetuses carrying a homozygous targeted mutation of microglobulin Made [Koller et al. (1990)Science  248: 1227-30]. Those feeder cells are G418 Can be grown in a medium containing. Trypsinize ES cells at 80% confluence. Thin treatment, concentration by simple centrifugation, and 2 x 10⁷HEPES buffer at a concentration of cells / ml Prepared for electroporation by resuspension in saline solution. The cell Was allowed to equilibrate at room temperature and linearized DNA (as described above) (20 μg) was added. . The mixture was run at 960 μF using a BioRad Gene Pulser. And electroporation at 250V. After leaving the cells at room temperature for 10 minutes, 4 × 10 plates of mitomycin C-treated feeder cells (3 × 10⁶Feeder cells / plate ) Applied over. After incubation for 48 hours at 37 ℃, 150 μg / ml (effective concentration) ) G418-containing medium was supplied to the cells.D. Analysis of ES cells with constant region targeting   After 7 to 10 days under drug selection using G418, each surviving individual colony was Harvest, dissociate in a drop of trypsin in a 96-well plate, then at 37 ° C. Incubated for 2 minutes. Mitomycin C treated cells from each colony Wells of a 24-well plate containing feeder cells and selection medium containing 150 μg / ml G418 Moved to. After an additional 5-8 days, freeze 20% of the cells in each well and Used for preparation of genomic DNA. 0.4 ml of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, Overnight with 10 mM EDTA, 1% SDS and proteinase K (1 mg / ml) at 50 ° C. The cells were lysed by incubating. Phenol extraction and ethanol DNA was purified by precipitation. Wash the DNA pellet with 70% ethanol and dry. It was dried and resuspended in TE (20 μl).   Southern analysis was performed using BglII digested genomic DNA from each sample . Original phage flanking the fragment used for 3'homology in the targeting vector When the 1.2 kb BamHI / BglII fragment isolated from DNA was used as a probe, A 2.3 kb fragment was detected from the ES cell locus, while targeted E A larger 4.9 kb fragment was detected from the S cell locus (Fig. 7). B when connecting BglII / BamH in the targeting plasmid due to the binding of glII and BamHI sites The BglII site in the I fragment was lost and a new one placed in the thymidine kinase gene. BglII site is targeted The size of the fragment was increased due to its introduction into the defined locus.   From the Southern analysis screen above, three different targeting pluses Of the 103 clones derived from the experiment using Mido, Five cell lines were identified (Table 1).   After thawing and propagating, four positive clones (clone 625, 604, 611 and Further analysis of genomic DNA prepared from S. 653) reconfirmed the first observations. It was As a second probe, a 1.7 kb HindIII / BglII fragment extending in the J region of the κ locus One piece is used for homologous targeting at the 5'end of the targeting vector. To find out the exact built-in pattern. EcoRI digestion of this probe and genomic DNA Was used to detect a 15 kb fragment from the unmutated allele. By contrast, target Thymidine kinase gene during homologous integration A 7.8 kb fragment was detected as a result of the introduction of a new EcoRI site in (Fig. 7).E. FIG. In vitro J region DNA from targeted clones Excision   Has the desired deletion from the homologously targeted kappa locus Cells from clone 653 were treated with ganciclovir (2 μM) and G418 (150 0.5-1 x 10 in the presence of both⁶Staining on feeder cells at a density of cells / 10 cm dish did. After growing for 5 days in the presence of both drugs, clones in 24 wells as above. Taken in plates and grown under G418 selection only. 5-8 more days later Freeze 20% of the cells in each well as described and use the rest for the preparation of genomic DNA. used.F. Analysis of ES cells lacking J / constant region   Southern analysis was performed using BamHI digested genomic DNA from each sample . The probe was the 0.8 kb EcoRI fragment used as ADH in the targeting vector. 12.7 kb fragment was detected from the native ES cell locus when used as ES cell gene with constant region targeted using DNA from sequence 653 A larger 15.9 kb fragment was detected from the locus (Fig. 7). Into the 12.7kb BamHI fragment Due to the insertion of the tk gene, ADH and neo gene, the size of the fragment was increased. ne o A new BamHI site was also introduced at the 3'end of the gene. J / Constant region deleted cells When used with these DNAs, they were targeted as predicted by Southern analysis. In addition to the 12.7 kb fragment from the unidentified allele, a 5.5 Kb fragment from the mutated locus was added. A piece was detected. 1.5 x 10 smeared⁶Obtained from individual ES cells (clone 653) From this screen by Southern analysis of two clones, the intended J and constant One cell line with a deletion of the region (clone 653B) was identified.   Further analysis of genomic DNA prepared from clone 653B after thawing and propagation Reaffirmed his first observations. Targeting target using 0.8 kb EcoRI fragment Two alternatives that would cut outside the excision region on the 5'and 3'ends of the Check for deletions by restriction digests It was Thus this probe and the BglII digest of genomic DNA from unexcised clone 653. , The 2.6 kb fragment was detected from both the unmuted and mutant alleles. An additional 4.9 kb fragment from only the targeted (Fig. 7). This 4.9 kb fragment is the same as the previous 1.2 kb BamHI / BglII fragment. It was the same as that detected. BglII when using DNA from clone 653B Digestion exposed the 5.8 kb fragment in addition to the 2.6 kb fragment from the unmutated allele. 0.8k b SacI digest of clone 653 DNA probed with EcoRI fragment is unmutated Shows 5.5 kb fragment from both allele and variant allele, and targets A 3.1 kb fragment was shown only from all the alleles (Fig. 7). From clone 653B The above 5.5 kb fragment and an additional 2.0 kb fragment were also detected in the DNA of. 5.8kb BglII disconnected The fragment and the 2.0 kb SacI fragment are consistent with the analysis of putative restriction maps for the precise excision step. It was In this case, a 10.3 kb DNA containing the J region, tk gene and one copy of ADH Was deleted.G. Generation of germline chimeras   Unmutated E14-1 cells are frequently germline after injection into C57BL / 6J blastocysts Contributed to. Cells from the targeted ES cell line 653B were generated as described above. Proliferated on feeder layers, trypsinized, then 15% fetal bovine serum, 20 mM H DMEM supplemented with EPES (pH 7.3), antibiotics and β-mercaptoethanol It was resuspended in an injection medium consisting of Inject ES cells (10-15) into each blastocyst and Transferred blastocysts (10) into pseudopregnant female mice and transfer 5 into each uterine horn It was The chimeric progeny are identified by the chimeric coat color. Crossing male chimera with C57BL / 6J female Of 129-derived ES cells (unmutated or targeted) Germline transmission is detected by the agouti coat color of F1 progeny.   All publications and patent applications mentioned in this specification are related to the invention to which they belong. Indicates the technical level of the worker. All publications and patent applications are as if they were individual publications. Or was it pointed out that the patent application is clearly and individually incorporated as a reference? As such, it is incorporated herein by reference. The present invention has been fully described above. There are numerous modifications to the invention without departing from the spirit or scope of the appended claims. And it will be apparent to those skilled in the art that modifications can be made.                             Sequence listing (1) General information:     (I) Applicants: Brenner, Daniel G.                  Dubridge, Robert B.                  Otten, Gillis R.    (Ii) Title of invention: Method for limiting deletion of DNA   (Iii) Number of sequences: 12    (Iv) Contact:          (A) Destination: Flehr, Hohbach, Test, Albritton & Herbert          (B) Street: Four Embaroadero Center, Suite 3400          (C) State: California          (D) Country: USA          (E) ZIP: 94111     (V) Computer readable format:          (A) Medium type: floppy disk          (B) Computer: IBM PC compatible type          (C) Drive system: PC-DOS / MS-DOS          (D) Software: PatentIn Release # 1.0,                              Version # 1.25    (Vi) Application data:          (A) Application number: PCT / US94 /          (B) Application date: March 11, 1994          (C) Classification:   (Viii) Agent information:          (A) Name: Roland, Bertram I.          (B) Registration number: 20,015          (C) Reference number: FR-57537 / BIRCELL-015    (Ix) Telecommunications information:          (A) Telephone: (415) 494-8700          (B) Telefax: (415) 494-8771          (C) Telex: 910 277299 (2) SEQ ID NO: 1     (1) Sequence features:          (A) Sequence length: 57 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..53          (D) Other information: complementary to bases 1 to 52 of SEQ ID NO: 2    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 2..7          (D) Other information: NdeI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 8..14          (D) Other information: Bsu36I restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 19..24          (D) Other information: SphI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 25..30          (D) Other information: KpnI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 31..36          (D) Other information: EcoRI restriction site    (Ix) Features:          (A) Characteristic symbol: misc-feature          (B) Location: 40..45          (D) Other information: HindIII restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 46..53          (D) Other information: NotI restriction site    (Xi) Sequence: SEQ ID NO: 1: (2) SEQ ID NO: 2:     (I) Sequence features:          (A) Sequence length: 56 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..52          (D) Other information: complementary to bases 1 to 52 of SEQ ID NO: 1    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..8          (D) Other information: NotI restriction site    (Ix) Features:          (A) Characteristic symbol: misc-feature          (B) Location: 9..14          (D) Other information: HindIII restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 18..23          (D) Other information: EcoRI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 24..29          (D) Other information: KpnI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 30..35          (D) Other information: SphI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 39..45          (D) Other information: Bsu36I restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 46..51          (D) Other information: NdeI restriction site    (Xi) Sequence: SEQ ID NO: 2: (2) SEQ ID NO: 3:     (I) Sequence features:          (A) Sequence length: 47 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..43          (D) Other information: complementary to bases 1-43 of SEQ ID NO: 4    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..6          (D) Other information: SacI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 7..12          (D) Other information: BamHI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 16..21          (D) Other information: XhoI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 22..27          (D) Other information: EcoRI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 31..36          (D) Other information: HindIII restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 37..42          (D) Other information: NdeI restriction site    (Xi) Sequence: SEQ ID NO: 3: (2) SEQ ID NO: 4:     (I) Sequence features:          (A) Sequence length: 47 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..43          (D) Other information: complementary to bases 1-43 of SEQ ID NO: 3    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 2..7          (D) Other information: NdeI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 8..13          (D) Other information: HindIII restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 17..22          (D) Other information: EcoRI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 23..28          (D) Other information: XhoI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 32..37          (D) Other information: BamHI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 38..43          (D) Other information: SacI restriction site    (Xi) Sequence: SEQ ID NO: 4: (2) SEQ ID NO: 5:     (I) Sequence features:          (A) Sequence length: 50 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..46          (D) Other information: complementary to bases 1 to 46 of SEQ ID NO: 6    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..6          (D) Other information: HindIII restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 10..15          (D) Other information: EcoRI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 16..21          (D) Other information: KpnI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 23..28          (D) Other information: BamHI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 30..35          (D) Other information: SacI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 39..46          (D) Other information: NotI restriction site    (Xi) Sequence: SEQ ID NO: 5: (2) SEQ ID NO: 6:     (I) Sequence features:          (A) Sequence length: 50 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..46          (D) Other information: complementary to bases 1 to 46 of SEQ ID NO: 5    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..8          (D) Other information: NotI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 12..17          (D) Other information: SacI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 19..24          (D) Other information: BamHI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 26..31          (D) Other information: KpnI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 32..37         (D) Other information: EcoRI restriction site    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 41..46          (D) Other information: HindIII restriction site    (Xi) Sequence: SEQ ID NO: 6: (2) SEQ ID NO: 7:     (I) Sequence features:          (A) Sequence length: 13 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 1..13          (D) Other information: complementary to bases 5 to 17 of SEQ ID NO: 8    (Xi) Sequence: SEQ ID NO: 7: (2) SEQ ID NO: 8:     (I) Sequence features:          (A) Sequence length: 21 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 5..17          (D) Other information: complementary to bases 1 to 13 of SEQ ID NO: 7    (Xi) Sequence: SEQ ID NO: 8: (2) SEQ ID NO: 9:     (I) Sequence features:          (A) Sequence length: 26 bases          (B) Sequence type: nucleic acid          (C) Number of chains: single chain          (D) Topology: linear    (Ii) Sequence type: cDNA    (Xi) Sequence: SEQ ID NO: 9: (2) SEQ ID NO: 10:     (I) Sequence features:          (A) Sequence length: 30 bases          (B) Sequence type: nucleic acid          (C) Number of chains: single chain          (D) Topology: linear    (Ii) Sequence type: cDNA    (Xi) Sequence: SEQ ID NO: 10: (2) SEQ ID NO: 11:     (I) Sequence features:          (A) Sequence length: 16 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 5..16          (D) Other information: complementary to bases 5 to 16 of SEQ ID NO: 12    (Xi) Sequence: SEQ ID NO: 11: (2) SEQ ID NO: 12:     (I) Sequence features:          (A) Sequence length: 16 base pairs          (B) Sequence type: nucleic acid          (C) Number of chains: double-stranded          (D) Topology: linear    (Ii) Sequence type: cDNA    (Ix) Features:          (A) Feature symbol: misc feature          (B) Location: 5..16          (D) Other information: complementary to bases 5 to 16 of SEQ ID NO: 11    (Xi) Sequence: SEQ ID NO: 12:

[Procedure of Amendment] Patent Law Article 184-8 [Date of submission] June 1, 1995 [Amendment] Claims 1. A method of introducing a limited deletion into a chromosomal target site in a host cell, comprising: (1) transforming a viable cell with a DNA targeting construct, wherein the construct comprises (A) two targets A target DNA sequence which is a DNA sequence and is homologous to the DNA sequence defining the target site and which is separated by the following first v-ADH sequence; (B) first additional DNA homology (v-ADH B) a first v-ADH sequence homologous to a first chromosomal ADH sequence (c-ADH) at a site located at a distance from the target site on the same chromosome; C) comprising at least one selectable marker, (2) selecting for transformed cells expressing said selectable marker, and (3) selecting for transformed cells lacking said selectable marker. Wherein homologous recombination occurs at the target site resulting in integration of the DNA construct, and intramolecularly between the first v-ADH sequence and the first c-ADH sequence. A method wherein recombination occurs to result in a chromosomal deletion between said target site and said first c-ADH site. 2. The method of claim 1, wherein the at least one selectable marker comprises one positive selectable marker and another negative selectable marker. 3. 3. The method of claim 2, wherein the positive selection marker is located between the negative selection marker and the v-ADH sequence on the DNA construct. 4. The method of claim 2, wherein the ADH sequence comprises a mammalian repetitive DNA sequence. 5. The DNA construct further comprises a second v-ADH sequence and a second negative selection marker, wherein the second v-ADH sequence is located on the same chromosome as the target site. Is homologous to the c-ADH sequence of the c-ADH site of, and the target site is located between the first c-ADH site and the second c-ADH site on the chromosome. The method described. 6. The DNA construct comprises an additional (1 + x) v-ADH sequence and an additional (1 + x) negative selection marker, wherein the (1 + x) v-ADH sequences are tandem. Each c-ADH is homologous to (1 + x) additional c-ADH sequences at the (1 + x) additional c-ADH sites located, on one side of the target site on the same chromosome. A sequence is adjacent to the next in tandem, where "x" is any number of additional DNA homologous sequences, whereby a contiguous deletion of chromosomal DNA adjacent to the target site is achieved. The method according to claim 2, wherein 7. 3. The method of claim 2, wherein the distance between the target site and the c-ADH site is about 500 bp to about 4000 kb in length. 8. The method according to claim 8, wherein a DNA sequence encoding a transcription enhancer and / or a silencer is included in the chromosomal sequence between the target site and the c-ADH sequence. 9. The method of claim 2, wherein the v-ADH sequence is homologous to one of the target sequences and differs by at least 1 bp. Ten. The method according to claim 1, wherein the marker is selected from the group consisting of HPRT minigene, neo gene, HSV thymidine kinase gene, hygromycin gene resistance and HPRT gene. 11. A method of introducing a deletion into a mammalian immunoglobulin locus in a host cell, the method comprising: (1) transforming a viable mammalian cell with a DNA construct, wherein the construct comprises (A) two A target DNA sequence which is homologous to the DNA sequence of the constant region of the mammalian immunoglobulin locus; (B) the target sequence is separated by the v-ADH sequence and is the v-ADH sequence. A v-ADH sequence homologous to a c-ADH sequence at a site located at a distance on the same chromosome 5'or 3'to the target site; (C) a positive selection marker; D) comprising a negative selection marker; (2) selecting for transformed cells expressing the positive selection marker; and (3) for transformed cells lacking the negative selection marker. Selecting, whereby homologous recombination occurs at the mammalian immunoglobulin locus resulting in integration of the DNA construct and in the chromosome between the v-ADH and c-ADH sequences. A method wherein intramolecular recombination occurs resulting in a chromosomal deletion between said target site and said c-ADH site. 12. 12. The method of claim 11, wherein the immunoglobulin loci are selected from the group consisting of immunoglobulin heavy chain and light chain loci. 13. 12. The method of claim 11, wherein the immunoglobulin loci are light chain loci. 14. 14. The method of claim 13, wherein the immunoglobulin locus is the kappa chain locus. 15. 14. The method of claim 13, wherein the immunoglobulin loci are lambda chain loci. 16． A DNA construct comprising: (A) two target DNA sequences homologous to a DNA sequence proximal to a chromosomal target site and separated by the following v-ADH sequence; (B) a v-ADH sequence which is homologous to a c-ADH sequence in a chromosomal region located apart from the target homologous DNA sequence on the same chromosome; (C) a marker for positive selection; and (D) ) A DNA construct comprising a marker for negative selection. 17. 17. The DNA construct of claim 16, wherein the v-ADH sequence comprises a mammalian repetitive DNA sequence. 18. The construct further comprises a second v-ADH DNA sequence and a second negative selection marker, wherein the second v-ADH sequence is located on the same chromosome as the target site. 17. The DNA of claim 16, which is homologous to the c-ADH sequence of a chromosomal site and wherein the target site is located on the chromosome between the first c-ADH site and the second c-ADH site. Composition. 19. 17. The DNA construct of claim 16, wherein the chromosomal sequence between the target site and the c-ADH sequence is about 500 bp to about 4000 kb in length. 20. 17. The DNA construct according to claim 16, wherein the chromosomal sequence between the target site and the first c-ADH sequence further comprises a DNA sequence encoding a transcription enhancer and / or silencer. twenty one. 17. The DNA construct according to claim 16, wherein the marker is selected from the group consisting of HPRT minigene, neo gene, HSV thymidine kinase gene, hygromycin resistance gene and HPRT gene. twenty two. A method for inactivating a target locus on a chromosome between a target site on the same chromosome and a chromosomal c-ADH DNA sequence located at a distance from the target site, comprising: (1) DNA composition To transform viable mammalian cells, wherein the DNA construct is (A) two target DNA sequences homologous to the DNA sequence of the target site on the chromosome and has the following v-ADH sequence: (B) the v-ADH sequence that is homologous to the c-ADH sequence of the chromosomal site located apart from the target site on the same chromosome; (C) comprising a marker for positive selection; and (D) comprising a marker for negative selection; (2) selecting for transformed cells expressing said positive selection marker; and (3) enabling said negative selection. Selecting for transformed cells lacking a marker, whereby homologous recombination occurs at the target site resulting in integration of the DNA construct, and the v-ADH and c-ADH sequences. A method wherein intramolecular recombination occurs between and results in a chromosomal deletion of at least a portion of the target locus between the target site and the c-ADH site such that the target locus is inactivated. twenty three. 23. The method of claim 22, wherein the target locus is MHC. twenty four. The target locus beta ₂ - is microglobulin subunit locus, A method according to claim 22. twenty five. A method for producing mammalian donor cells lacking MHC class I and / or class II antigens for use in transplantation, comprising: (1) a DNA construct for inactivating class I and / or class II MHC antigens. To transform viable mammalian cells, wherein the DNA construct is (A) two target DNA sequences homologous to the DNA sequences in MHC and separated by the following v-ADH sequence: (B) a v-ADH sequence homologous to a c-ADH sequence at a site located at a distance on the 5'or 3'side of the MHC on the same chromosome. The v-ADH sequence; (C) a positive selection marker; and (D) a negative selection marker; (2) selecting for transformed cells expressing the positive selection marker; 3) selecting for transformed cells lacking the negative selectable marker, whereby homologous recombination occurs in the MHC resulting in integration of the DNA construct, and v- in the chromosome. Method for inactivating class I and / or class II MHC antigens by causing intramolecular recombination between the ADH sequence and the c-ADH sequence resulting in a chromosomal deletion between the target site and the c-ADH site . 26. The chromosomal deletion beta ₂ - comprising microglobulin locus, A method according to claim 2 5. 27. A normal mammalian cell that does not express a surface MHC antigen produced by the method of claim 25. 28. 28. The normal mammalian cell of claim 27, wherein the cell is a mouse. 29. 28. The normal mammalian cell of claim 27, wherein the cell is human.

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Otten, Gillis Earl.             United States of America, California 94404,             Foster City, Foster City               Boulevard 1183

Claims (1)

[Claims] 1. A method of introducing a limited deletion into a chromosomal target site in a host cell, comprising: (1) transforming a viable cell with a DNA targeting construct, wherein the construct comprises (A) two targets A DNA sequence which is homologous to the DNA sequence defining the target site and which is separated by the first v-ADH sequence below, (B) a first additional DNA homology (v- ADH) sequence, said first v-ADH sequence being homologous to the first chromosomal ADH sequence (c-ADH) of a site located at a distance from said target site on the same chromosome, and (C) comprising at least one selectable marker; (2) selecting for transformed cells expressing said selectable marker; and (2) selecting for transformed cells lacking said selectable marker. Wherein homologous recombination occurs at the target site resulting in integration of the DNA construct and a molecule between the first v-ADH sequence and the first c-ADH sequence. A method wherein internal recombination occurs resulting in a chromosomal deletion between said target site and said first c-ADH site. 2. 2. The method of claim 1, wherein the at least one selectable marker comprises one positive selection marker and another negative selection marker. 3. 3. The method of claim 2, wherein the positive selection marker is located between the negative selection marker and the v-ADH sequence on the DNA construct. 4. The method of claim 2, wherein the ADH sequence comprises a mammalian repetitive DNA sequence. 5. The DNA construct further comprises a second v-ADH sequence and a second negative selection marker, wherein the second v-ADH sequence is located on the same chromosome as the target site. Is homologous to the c-ADH sequence of the c-ADH site of, and the target site is located between the first c-ADH site and the second c-ADH site on the chromosome. The method described. 6. The DNA construct comprises (1 + x) additional v-ADH sequences and (1 + x) negative selection markers, wherein the (1 + x) v-ADH sequences are tandem. Homologous to the (1 + x) additional c-ADH sequences at the (1 + x) additional c-ADH sites located, each c-on one side of the target site on the same chromosome The ADH sequence is flanked by the next in tandem, where "x" is any number of additional DNA homologous sequences, thereby achieving a contiguous deletion of chromosomal DNA flanking the target site. The method of claim 2, wherein the method is performed. 7. 3. The method of claim 2, wherein the distance between the target site and the c-ADH site is about 500 bp to about 4000 kb in length. 8. The method according to claim 8, wherein a DNA sequence encoding a transcription enhancer and / or a silencer is included in the chromosomal sequence between the target site and the v-ADH sequence. 9. The method of claim 2, wherein the v-ADH sequence is homologous to one of the target sequences and differs by at least 1 bp. Ten. The method according to claim 1, wherein the marker is selected from the group consisting of HPRT minigene, neo gene, HSV thymidine kinase gene, hygromycin resistance and HPRT. 11. A method of introducing a deletion into a mammalian immunoglobulin locus in a host cell, comprising: (1) transforming a viable mammalian cell with a DNA targeting construct, wherein the construct is (A) 2. One target DNA sequence, said target sequence being homologous to the DNA sequence of the constant region of said mammalian immunoglobulin locus, (B) said target sequence being separated by a v-ADH sequence, And a v-ADH sequence homologous to a partial c-ADH sequence located at a certain distance 5'or 3'to the target site on the same chromosome; (C) a positive selection marker; And (D) comprising a negative selectable marker; (2) selecting for transformed cells expressing said positive selectable marker; and (3) lacking said negative selectable marker. Selecting for the transformed cells, whereby homologous recombination occurs at the mammalian immunoglobulin locus resulting in integration of the DNA construct, and at the chromosome the v-ADH sequence and the c -A method wherein intramolecular recombination occurs between ADH sequences resulting in a chromosomal deletion between said target site and said c-ADH site. 12. 12. The method of claim 11, wherein the immunoglobulin loci are selected from the group consisting of immunoglobulin heavy chain and light chain loci. 13. 12. The method of claim 11, wherein the immunoglobulin loci are light chain loci. 14. 14. The method of claim 13, wherein the immunoglobulin locus is the kappa chain locus. 15. 14. The method of claim 13, wherein the immunoglobulin loci are lambda chain loci. 16． A DNA construct comprising: (A) two target DNA sequences homologous to a DNA sequence proximal to a chromosomal target site and separated by the following v-ADH sequence; (B) a v-ADH sequence which is homologous to a c-ADH sequence in a chromosomal region located apart from the target homologous DNA sequence on the same chromosome; (C) a marker for positive selection; and (D) ) A DNA construct comprising a marker for negative selection. 17. 17. The DNA construct of claim 16, wherein the v-ADH sequence comprises a mammalian repetitive DNA sequence. 18. The construct further comprises a second DNA v-ADH sequence and a second negative selection marker, wherein the second v-ADH sequence is located on the same chromosome as the target site. 17. The DNA of claim 16, which is homologous to the c-ADH sequence of a chromosomal site and wherein the target site is located on the chromosome between the first c-ADH site and the second c-ADH site. Composition. 19. 17. The DNA construct of claim 16, wherein the chromosomal sequence between the target site and the c-ADH sequence is about 500 bp to about 4000 kb in length. 20. 17. The DNA construct according to claim 16, wherein the chromosomal sequence between the target site and the first v-ADH sequence further comprises a DNA sequence encoding a transcription enhancer and / or silencer. twenty one. 17. The DNA construct according to claim 16, wherein the marker is selected from the group consisting of HPRT minigene, neo gene, HSV thymidine kinase gene, hygromycin resistance and HPRT. twenty two. A method for inactivating a target locus on a chromosome between a target site on the same chromosome and a chromosomal c-ADH DNA sequence located at a distance from the target site, comprising: (1) DNA The construct is used to transform a viable mammalian cell, wherein the DNA construct is (A) two target DNA sequences homologous to a DNA sequence at a target site on a chromosome, and the following v-ADH The target sequence separated by a sequence; (B) the v-ADH sequence, which is homologous to the c-ADH sequence of a chromosomal site located away from the target site on the same chromosome; (C) a marker for positive selection; and (D) a marker for negative selection; (2) selecting for transformed cells expressing the positive selection marker; and (3) allowing the negative selection. Selecting for transformed cells lacking a functional marker, whereby homologous recombination occurs at the target site resulting in integration of the DNA construct, and the v-ADH sequence and the c-ADH. A method wherein intramolecular recombination between sequences occurs resulting in a chromosomal deletion of at least a portion of the target locus between the target site and the c-ADH site, and the target locus is inactivated. twenty three. 23. The method of claim 22, wherein the target locus is MHC. twenty four. The target locus beta ₂ - is microglobulin subunit locus, A method according to claim 22. twenty five. A method for producing a mammalian donor cell lacking MHC class I and / or class II antigen for use in transplantation, comprising: (1) a DNA construct for inactivating a class I and / or class II MHC antigen. Is used to transform viable mammalian cells, wherein the DNA construct is (A) two target DNA sequences homologous to the DNA sequences in MHC, separated by the v-ADH sequence. (B) the v-ADH sequence, which is homologous to the c-ADH sequence of a site located at a certain distance on the same chromosome on the 5'or 3'side of the MHC. An ADH sequence; (C) a positive selection marker; and (D) a negative selection marker; (2) selecting for transformed cells expressing said positive selection marker; and (3) Selecting for transformed cells lacking the negative selectable marker whereby homologous recombination occurs in the MHC resulting in integration of the DNA construct and the v-ADH in the chromosome. A method for inactivating a class I and / or class II MHC antigen by causing intramolecular recombination between a sequence and said c-ADH sequence resulting in a chromosomal deletion between said target site and said c-ADH site. 26. The chromosomal deletion beta ₂ - comprising microglobulin locus, A method according to claim 25. 27. A mammalian cell that does not express a surface MHC antigen produced by the method of claim 25. 28. 28. The mammalian cell of claim 27, wherein the cell is a mouse. 29. 28. The mammalian cell of claim 27, wherein the cell is human.