METHOD OF EXCISING AND PROPAGATING GENOMIC DNA

Abstract: A process for isolating, cloning and maintaining large genomic fragments of chromosomal DNA. This process includes the steps of introducing a marker into the chromosomal DNA to be cloned, inserting double-strand breaks into the chromosome followed immediately by combining the cells containing the chromosome with the fragmented marker to another cell line capable of harboring extrachromosomal DNA elements, and subsequently isolating genomic fragments by separating the extrachromosomal DNA from the chromosomal DNA of the other cell line.

Description: METHOD OF EXCISING AND PROPAGATING G.E.NOMIC DNA Cross-Reference to Related Application This application is a continuatiόn-ih-p rt αf an earlier U.S. application. Serial No, 07/413,851, filed Septer ->er 28, 1989. Background of the Invention This invention relates to a method for* the isolation, cloning and maintenance of large chromosomal fragments of deoxyribonucleic acid (DNA) . The defining of DNA sequences over large chromosomal region© appears to promise a greater understanding of ttøe biochemical and physiological mechanisms of human gene regulation. The haploid human genome (one set of the two sets of chromosomes) is estimated to be three billion base- pa:.-3 in length. Many restriction fragment length polymorphism (RFLP) marker sequences have been located genetically on the human genomic map, and the distance between these markers is in the 1 - 5 centimorgan range. This is a genetic distance based on crosβover frequency, but a centimorgan corresponds roughly to a million base pairs in physical length. The chromosomal location of many human genes with respect to these RFLP markers is known. However, this knowledge of the approximate location of these genes is of limited utility because it has not been possible to isolate, clone, and maintain DNA sufficiently long such that the RFLP markers are physically associated with the gene of interest. Standard methods of cloning and isolating DNA sequences have been limited to 50 thousand base-pairs (kb) in length, the length of DNA that will fit into a lambda phage for cosmid cloning. Newer techniques based on construction of artificial chromosomes in yeast are capable of maintaining DNA much longer in length. However this technique is limited in that the DNA must be purified in solution prior to insertion in yeast. This limits the maximum length of the yeast artificial chromosome to the length of DNA in solution which is in turn limited by the shearing effects of liquid. In practice this limits the DNA size to no greater than several hundred kb. Genes located more than several hundred kb from RFLP markers will not be physically associated with them, so these markers cannot be easily used to identify and recover those genes. A need thus exists for a method of isolating, cloning and maintaining DNA fragments that prevents shearing from taking place and produces longer chromosomal fragments than heretofore available. Summary of the Invention This need is satisfied and other benefits realized, in accordance with the present invention by: introducing a marker into a chromosome from a first cell line; inserting double-strand breaks into the chromosome to produce genomic fragments of DNA; and fusing the cells containing the chromosomal fragment having the marker to cells from a second cell line which are capable of " harboring chromosomal fragments as extrachromosomal DNA, thereby preventing DNA from being by itself in solution. ' Large fragments of DNA containing the marker are finally isolated by separating the extrachromosomal DNA fϊ*om the chromosomal DNA of the second cell line. By averting the shearing effect, this process facilitates the mapping and recovering of important genes where the chromosomal location is imprecisely known In accordance with one embodiment of this invention, the marker introduced into the DNA of a chromosome to be cloned is, preferably, dominant, selectable and amplifiable such that only one copy d the marker is needed (th dominant aspect , cells containing chromosomes trit are harboring the marker can be recovered (the selectable aspect) , and cells containing extra copies of the marker cgm be selected (the amplifiable aspect) . In accordance with another aspect of the present invention, the extrachromosomal DNA is separated from the chromosomal DNA of the second cell line on the basis of size differences between extraόhromoso&al DNA and standard chromosomes, i.e., by ftadtionating "the total DNA by size. Preferably, this is accomplished bf, but not limited to, using either sucrose gradients or g^l electrophoresis. Brief Description of the Drawings These and other features, aspects and advantag of the present invention will be more readily understood from the following detailed description when read in conjunction with the accompanying drawing' figure which schematically illustrates the method of the presentation. Description of the Preferred Embodiment The method of the instant invention can be advantageously employed to isolate, clone and maintain DNA fragments that are large enough to allow for precise mapping of gene segments on the chromosome. The method should be particularly useful for cloning large pieces of human DNA, but in principal, it can be useful for cloning large pieces of DNA from any species. The products of this process are large defined regions of human genomic DNA maintained in a non-human (generally mouse cell) background. One principal utility of the process is the production of genomic libraries suitable for mapping and cloning genes. The process will serve to bridge a gap between the whole genome -or human chromosome specific somatic cell hybrids - and current cloning techniques for genomic library construction. The presently available techniques are limited to an upper size of approximately 50 thousand base pairs for cosmid clones, and several hundred thousand basepairs for yeast artificial chromosomes whereas the smallest human chromosome is approximately 100 million basepairs. These techniques are limited by, among other factors, the required purification of the DNA which introduces shearing of the DNA molecules. The method may be used as an aid to mapping, identifying, and recovering important genes located on isolated regions, or to isolate chromosome region- specific probes for diagnosis of human chromosome- rearrangement disorders. An additional benefit will be that these regions of DNA should direct the synthesis of proteins whose genes reside on these DNA regions. Therefore, this process should be useful for recovering the gene products (proteins) as well as the genes themselves. The amplification of the inserted sequences attached to the amplifiable marker sequences will frequently lead to overproduction of the proteins located on the inserted foreign (generally human) DNA. This will aid in the identification of the proteins located on these regions, and as a by-product may facilitate commerical production. The method generally involves the following steps, which are labeled on the accompanying drawing and described in greater detail in the examples provided below. Step 1: A mammalian cell line is initially selected whose cells contain chromosomal DNA that one wishes to clone. This cell line is referred to as the donor cell line (10) . A second mammalian cell line, referred to as the recipient cell line (14) , is also selected based on its previously demonstrated ability to maintain large chromosomal fragments, .preferably a__f* extrachromosomal double minutes ( ahl G.M. , Cancer Research, vol. 49 p. 1333 (1989)). For example, maiise EMT-6 cells readily harbor double minute chromosomes. Step 2: A marker (18) is inserted into one chromosome (12 1 ) of chromosomes (12) αf the donor cell (10) to be cloned. Marker (18) includes & dominant suitable marker physically and contiguously attached to an amplifiable marker. In some cases the ominan marker may itself be amplifiable, as would be the case where the dihydrofolate reductase (DHFR) gene is used as the dominant marker and the recipient cell has no -6- functional dihydrofolate reductase. The method of introduction can be by any of several established methods such as DNA-mediated gene transfer by co-transfection of markers carried on bacterial 5 plasmids (Wigler, Silverstein, Lee, Pellicer, Cheng, and Axel, Cell, vol. 11, pp. 223-232 (1977)) or by defective retroviral insertion of linked genes (Cepko, Roberts, and Mulligan, Cell, vol. 37, p. 1053 (1984)). Step 3. Double-strand breaks (20) are introduced 10 into the chromosomes (12) of the donor cell (10) , for example, by lethal ionizing irradiation (Cirullo, Dana, Wasmoth, Mol. Cell. Biol., vol. 3 pp. 892-902 (1983)) . Step 4: The donor cells (10) containing the broken chromosomes are fused to the recipient cells (14) by any 15 of the standard protocols (Norwood and Zeigler, Techniques in Somatic Cell Genetics, Plennum Press, New York, pp. 35-45 (1982)). Known fusing agents, "fusogens", such as polyethylene glycol and dimethyl sulfoxide are added to a mixture of the two cell types 20 to increase the rate of cell membrane coalescence. This treatment causes the formation of hybrid cells containing both the broken chromosomes of the donor cell (10) , and the unbroken chromosomes of the recipient cell (14). 25 Step 5: The cells from the fusion treatment are placed under selective conditions such that only hybrid donor-recipient cells containing the selectable marker (18) will survive (Hahn, Morgan, and Painter, Somatic Cell and Mol. Gen., vol. 13 pp. 597-608 (1987)). For 30 instance, if the dominant selectable marker is the Neo gene which confers resistance to the aminoglycoside antibiotic G418, the cells are placed in medium containing sufficient G418 to kill all cells not containing the Neo gene. (The amount must be determined for each cell line but is generally in the 200 - 400 μg/ml active ingredient range.) Since the non- hybridized recipient cells do not contain this gene, and ' the donor cells containing the gene have all been lethally irradiated, the only survivors will be cells that are fusions between the two types. Step 6. If the marker (18) is susceptible to selection for gene amplification (increase in gene copy number) , or is physically associated with a gene susceptible to selection for amplification (Ringold, Dieckman, and Lee, J. Mol. Appl. Gen., vol. 1, pp. 165- 175 (1981)), then the number of extrachromosomal elements (12') per cell can be increased, by methods known in the art. Such methods are based on the observation that a few cells in a population will contain extra copies of a gene and that these cells produce more of a gene product. Any selection for overproduction such as resistance to higher levels of a toxic agent such as methotrexate to select for increased dihydrofolate reductase levels, will cause those cells containing the extra copies of the gene to grow and dominate the population at the expense of the cells with fewer copies. Step 7: The extrachromosomal elements (12') are then separated from the chromosomal DNA by fractionating the total DNA by size, either by sucrose density gradient centrifugation (Kaufman, Brown, and Schimke, PNAS USA, vol. 76, pp. 5669-5673 (1979)), or pulsed- field gel electrophoresis (Schwartz and Cantor, Cell, vol. 37 p. 67 (1986)). Since the extrachromosomal elements are much smaller than chromosomes, size fractionation results in effective separation and recovery of the DNA fragment derived from the donor cell (10), i.e., the fragment containing the selective marker (18) from the recipient cell's (14) chromosomal DNA. Using the above described process, chromosomal fragments of DNA thousands of kilobase pairs in length, e.g., one thousand to three thousand kilobase pairs, have been effectively isolated, cloned and maintained. It is anticipated that larger chromosomal fragments of DNA, on the order of 10 megabase pairs, will be isolated in the future. Specific examples of this new method will now be presented. EXAMPLE 1 Step 1: The donor cell (10) selected was a CHO (Chinese hamster ovary) cell line DG 44. Both copies of the DHFR gene were deleted from the chromosomes of this cell line by x-ray mutagenesis (Urlaub, Kas, Carothers and Chasin, Cell, vol. 33 pp. 405-412 (1983)) making it ideal for introducing both a Neo resistance gene and a DHFR gene as markers. The recipient cell (14) selected was a mouse EMT-6 cell line. This cell line was chosen because the line harbors double minute chromosomes in the 1-10 million base pair range. The dominant selectable marker (18) selected was the Neo gene, which, as is well known in the field, confers resistance to the antibiotic G418. Step 2: The two markers were introduced by co- transfection (Wigler, Silverstein, Lee, Pellicer, Cheng and Axel, Cell, vol. 11, pp. 223-232 (1977)), which results in the co-integration of two different plasmids in the same location in a mammalian chromosome. One million DG 44 cells were exposed to a calcium phosphate precipitate of 10 μg of plasmid DNA (5:1 ratio of PSV2DHFR to pZipNEO) for four hours. The media was then changed, and the cells exposed for 3 minutes to a 10% DMSO solution. After two days, the cells * were exposed to media lacking glycine, hypoxanthene and thymidine, and supplemented with 300μg/ml of G418 to select for cells that had received both markers. One of the numerous surviving colonies was arbitrarily chosen as an example to be subject to the fragment transfer procedure. Cells from this colony were expanded in number to ten million cells. Step 3: In order to introduce chromosome fragments from the donor cells (10) into the recipient cells (14) , two million donor cells that received the linked Neo and DHFR markers were subjected to 50 or 100 Gy. irradiation by a 320 Kilovolt Phillips industrial irradiator in order to fragment the chromosomes. Step 4: Five hundred thousand X-rayed donor cells and one hundred thousand recipient cells in five milliliters of Hams F12 medium with 10% fetal calf serum were placed into each of three 25 cm 2 flasks. Three hours were allowed for attachment of the cells to- the flask. Each flask was then rinsed three times with two milliliters of Hams F12 medium with fusogen (50% polyethylene glycol, 10% dimethyl sulfoxide (DMSO) in Hams F12) for one minute. The flasks were then rinsed three times with 10% DMSO in Hams F12 and twice with 10% fetal calf serum in Hams F12. The cells were allowed to recover in 10% fetal calf serum in Hams F12 medium. Step 5: To select hybrid donor/recipient cells, the fused cells were allowed two days under non- selective conditions to permit expression of tJie G418 resistance gene, and then plated at one hundred thousand cells per 100 ran dish in 10% fetal calf serum in Hams F12 with G418, at a G418 concentration of 400 μg/ml. After three weeks, five colonies were selected for further analysis. These colonies were individually isolated by poking a pasteur pipette with trypsin/EDTA (by dipping the tip of the upright pasteur pipette into the trypsin/EDTA solution used for trypsinizing the cells) into the center of the colony to be selected and expelling the cells into a well of a 24 well plate. The populations of cells from individual colonies were expanded in number, by growing in the presence of 400 μg/ml of G418 and 10% fetal calf serum in Hams F12. Step 6: When the populations reached one million cells, each population was subdivided into two groups. One group was expanded for analysis of DNA, and the other grown in the presence of incremental increases in MTX, starting with 150nM MTX. When the cells could grow in that concentration of MTX, the MTX level was doubled. The process was repeated until the cells could grow in 1200 nM MTX. At this point, the level was increased to 1500 nM MTX and the DNA was prepared for analysis by embedding the cells in agarose at one million and ten million cells per 200 μl, and then digesting the cells in 1% Proteinase K, 10% Sodium Dodecyl Sulfate and 0.5M EDTA, for 24 hours at 55°C. Step 7: Isolation of the extrachromosomal elements was accomplished by using gel eleσtrophoresis of DNA from cells immobilized in agarose "plugs" that had been subjected to 20 - 50 Gy. X-irradiation immediately prior to electrophoresis, in order to linearize circular molecules. Measurable fragments were obtained that were 1, 1.5, and 3,000 kilobase pairs as determined by reference to yeast chromosomal markers. EXAMPLE 2 The purpose of this variant of the process is to produce mouse cell lines that contain double minute chromosome fragments derived from a specific human chromosome (chromosome 17) . Step 1: The donor cell line chosen was a mouse cell line harboring a single human chromosome 17 (LI cells from E. Stubblefield, M.D. Anderson). The receipient cell lir <s was mouse EMT-6. Step 2: The dominant marker chosen was the Neo gene on the defective retrovirus pZipNeo (Cepko, Roberts, and Mulligan, Cell, vol. 37, p. 1053 (1984)). To introduce this marker, we utilized the packaging cell line PA317 (American Type Culture Collection #CRL9078) which had been previously transfected with the plasmid pZipNeo. This cell line is capable of packaging RNA produced by the tranfected plasmid sequences into defective retroviral particles that in turn are excreted into the media in which the transfected PA317 cell are growing. These particles lead to high rates of infection and expected random integration of DNA produced from the infective RNA when the media from the pZipNeo containing PA317 cells is placed in contact with the donor cells. Step 3: The LI cells were infected by replacing their media with media that had been used to culture PA317 pZipNeo cells for 12 to 24 hours. LI cells growing in 74 cm 2 flasks were infected 3 consecutive times at 8-12 hour intervals, and then placed under selective conditions of 400 μg/ml G418 for 12-21 days. Step 4: The resulting G418 resistant cells in a confluent 75 cm 2 flask were subject to 200 Gy. X- irradiation. Step 5: The irradiated LI cells were fused to the EMT 6 cells at a ratio of approximately 5:1 donor to recipient cells, and a total of 10 5 recipients cells. Step 6: The fused cells were cultured in media containing 400 μg/ml G418 for 14-21 days. Approximately 350 colonies emerged and were individually isolated. Step 7: Samples of each of the isolates were probed for the presence of human specific sequences by attaching to nylon membranes (Abbott, Poisz, Byrne, Kwok, Sninsky, and Drlich, J. Inf. Dis., vol. 158, pp. 1158-1169 (1988)), lysing in situ (Rossman and Lubin, Som. Cell Mol Gen., vol. 14, p. 321 (1988)), and probing for the presence of the Alu repeat-sequence DNA (Gusella, Keys, Varsanyi-Breiner, Kao, Jones, Puck and Housman, "Isolation and localization of DNA Segments from Specific Chromosomes", Proc. Natl. Acad. Sci. USA, vol. 77, pp. 2829-2833). Step 8: Approximately 35 isolates were positive for the presence of the human repeat sequence. Some of these isolates contain the pZipNeo marker and the human Alu sequence on the same sized Not 1 fragment (800 kbp) as judged by Southern hybridization of pulsed-field gel electrophoretic separation of Not 1 digested DNA from the isolates. The membranes were probed sequentially with probes for the G418 resistance gene and then the Human Alu repeat DNA. The chromosomal location of these sequences can be determined by several procedures including in-situ hybridization of the DNA to metaphase arrested human chromosomes. In principal, the processes described herein for creating double minute chromosomes could be applied to cloning and mapping the genomes of any eukaryotic organism. However, several factors would have to be taken into consideration. The selections of the various markers will depend on the recipient cell, the donor cell and the objective of the "cloning". Genes that function in animal cells may not function in plant cells without modification. For instance, the 418 resistance gene functions in plants and insects as well as mammalian cells, but the specific plasmid constructions described in the presented examples would probably not function in most plants or insects because transcriptional control signals differ between these groups. These genes function only when they are genetically engineered into plasmids that contain functioning transcriptional signals appropriate for each organism. For Drosophyla melanogaster, the P element construction pϋChsneo (Stellar, and Pirrotta, EMBO, vol. 4 p. 167, (1985)) functions much like the pZipneo plasmid functions in mammalian cells in that it can lead to both random integration and G418 resistance. The 35S CaMV promoter functions in most plants (Fro m et al., PNAS USA, vol. 82, pp. 5824-5828, (1985)). The method of introduction of the DNA into the cells of different types of organisms will also depend on the organism into which the DNA is being introduced. DNA can be introduced into plants via the agrobacterium Ti plasmid, electroporation (Fromm et al, PNAS USA, vol, 82. pp. 5824-5828 (1985)), or by "shotgunning". Cell fusion techniques will also vary depending on the donor and recipient cells. It may be possible to use this process to clone plant genomic DNA in mammalian cells, but the fusion technique will be, very different since plant cells have cell walls and a central vacuole used for metabolic waste storage. However, nucleii can be readily isolated from plant tissue, and these organs should fuse readily with mammalian cells. From the foregoing description, it will be appreciated that a method of isolating, cloning and maintaining large DNA fragments has been developed which will facilitate the mapping and recovering of important genes where the chromosomal location is imprecisely known. Although specific embodiments have been described in detail, it will be apparent to those skilled in this art that various modifications, substitutions, additions and the like can be made without departing from the spirit of the invention. For example, other known markers, recipient and donor cells, introduction, fusion, selection, amplification and isolation approaches may be employed. All such equivalents are intended to be encompassed within the scope of the invention, as defined by the appended claims.

Claims: What is claimed is: 1. A process for the isolation and maintenance of large genomic fragments of chromosomal DNA which comprises the steps of: (a) introducing a marker into a chromosome from a first cell line; (b) inserting double-strand breaks into . the chromosome to produce genomic fragments of DNA, followed by fusion of the cell containing the chromosomal fragment having the marker with a second cell line, said chromosomal DNA of said second cell line being capable of harboring said chromosomal fragment from said first cell line as extrachromosomal DNA; and (c) isolating genomic DNA fragments containing said marker by separating the extrachromosomal DNA from the chromosomal DNA of said second cell line. 2. A process according to claim 1 wherein the marker is dominant, selectable and amplifiable. . 3. A process according to claim 1 wherein the marker comprises a dominant selectable marker and an amplifiable marker physically and contiguously attached to the dominant selectable marker. 4. A process according to cl £m 3 wherein the . marker comprises a combination of the gene for neomycin phosphotransferase and the dihydrofolate reductase gene. 5. A process according to claim 3 further comprising the steps of: selecting only hybrid second cells containing the fused extrachromosomal DNA; and amplifying the number of fused extrachromosomal DNA contained within said hybrid second cells. • 16- 6. A process according to claim l wherein mouse cells are used for said second cell line capable of harboring extrachromosomal DNA. 7. A process according to claim 1 wherein said step of fusing the chromosomal fragment containing said marker to the chromosomal DNA of a second cell line is accomplished immediately after the insertion of double- strand breaks into the chromosome of the first cell line to produce said genomic fragments of DNA. 8. A process according to claim 1 wherein the extrachromosomal DNA is separated from the chromosomal DNA of said second cell line by fractionating the total DNA by size. 9. A process according to claim 8 wherein gel electrophoresis is used to separate the extrachromosomal DNA from the chromosomal DNA of said second cell line. 10. A process according to claim 8 wherein sucrose gradients are used to separate the extrachromosomal DNA from the chromosomal DNA of said second cell line. 11. A process according to claim 1 wherein said step of inserting double-strand breaks into the chromosome to produce said genomic fragments of DNA followed by fusion of the chromosomal fragment containing the marker of the chromosomal DNA of a second cell line includes exposing the chromosome to ionizing radiation. 12. A process according to claim 1 wherein G-418 Sulfate and methotrexate are used to select for the chromosome containing the marker. 13. A process according to claim 1 wherein G-418 Sulfate is used to select for the chromosome containing the marker. 14. A process for the isolation and maintenance of large genomic fragments of chromosomal DNA, said process comprising the steps of: (a) selecting a first cell line containing chromosomal DNA to be isolated, and a second cell line, selected based on its ability to maintain large chromosomal fragments; (b) inserting a marker into one of the chromosomes of the first cell line, said marker comprising a dominant selectable marker physically and contiguously attached to an amplifiable marker; (c) introducing double-strand breads into the chromosomes of the first cell line to produce genomic fragments of DNA; (d) fusing the cells of the first cell line to the cells of the second cell line, thereby producing hybrid cells containing both the chromosomal fragments of the first cell and the chromosomes of the second cell; (e) selecting only hybrid cells of said first cell line - second cell line containing the marker; (f) amplifying the number of extrachromosomal DNA per hybrid cell; and (g) separating the extrachromosomal DNA from the chromosomal DNA of the hybrid cells by fractionating the total DNA by size.

Inventors: HAHN PETER J, LANE MICHAEL J, HAHN, PETER, J., LANE, MICHAEL, J.
Applicants: UNIV NEW YORK, THE RESEARCH FOUNDATION OF STATE UNIVERSITY OF NEW
Application year: 1990
Main classification: C12N5
IPC codes: C07K14, C12N15