DNA SEQUENCE IN THE Sp5 MUTANT AND SITE SPECIFIC GENETIC RECOMBINATION

Abstract: This invention relates to a DNA sequence of 956 base pairs as given in Figure 2, sequence list 1 and to a DNA sequence including two repetitive sequences consisting of ATTGATTAT and ATTATTAT as repeat units separated by a spacer of one base pair at 5' end and of two TGTATAATA as repeat units separated by a spacer of one base pair at 3' end. It also relates to methods for a site specific recombinogenic process, to a method for introducing a gene of interest into mammalian cells and a method for removal of sequences previously introduced by any gene targeting method. The use of 5'-TCTTT T TCTTT-3' for recombination in mammalian cells and the use of a direct repeat sequence of 10 bases and one base spacer, 5'-TCTTT T TCTTT-3' for transgenic work and as for a tool of site specific recombination are also claimed.

Description: DNA sequence in the Sp5 mutant and site specific genetic recombination This invention relates to a DNA sequence of 956 base pairs as given in Figure 2, sequence list 1 and to a DNA sequence including two repetitive sequences consisting of ATTGATTAT and ATT ATT AT as repeat units separated by a spacer of one base pair at 5 ' end and of two TGTATAATA as repeat units separated by a spacer of one base pair at 3 ' end. It also relates to method for a site specific recombinogenic process, to a method for introducing a gene of interest into mammalian cells and a method for removal of sequences previously introduced by any gene targeting method. The use of 5 '-TCTTT T TCTTT - 3' for recombination in mammalian cells and the use of a direct repeat sequence of 10 bases and one base spacer, 5 '-TCTTT T TCTTT - 3' for transgenic work and as for a tool of site specific recombination are also claimed. Introduction The invention is based on specific repeat sequences to be recognized by endogenously expressed mammalian protein(s) to execute a novel type of site-specific recombination in mammalian cells to be used as a complementary tool in genetic engineering. Prior art Different types of genetic recombination have been described, e.g., homologous, illegitimate, site-specific and V(D)J processes, as well as the transposon-mediated type. In some cases the mechanisms involved have been successfully explored in detail at the molecular level, more recently even in mammalian cells. V(D)J recombination is a process that assembles immunoglobin (Ig) and T cell receptor (TRC) genes during the development of lymphoid cells. (Gellert M, Annu. Rev. Genet 1992:22, 425-46; Van Gent et al, Cell 1996:85, 107-113). McBlane et al. (1995) revealed the roles played by the RAGl and RAG2 proteins in connection with V(D)J recombination. The mechanisms underlying this process is site-specific in the sense that it involves recognition site sequences (RSS), but the recombination event itself involves junctional flexibility. The V(D)J recombination mechanism has only been revealed in lymphatic cells. An endogenous site-specific recombination mechanisms in mammalian cells which results in exact junctions as has been described for recombination in lower organisms has not yet been reported and is the mechanistic basis of the invention. The knowledge of site-specific recombination resulting in exact junctions refers to studies in bacteria and yeast (Stark et al., 1992). These processes have been classified on the basis of their different mechanisms of action into two families, e.g., the λ integrase and the Tn3 resolvase families (Stark et al., 1992). In mammalian cells exact junctions can be produced by constructing site-specific recombination involving Cre-LoxP of the λ integrase family (Smith et al., 1995, Ramirez-Solis et al, 1995), which have been used extensively as tools for genomic engineering (Kilby et al., 1993). Several transgenic mouse strains have been successfully obtained using site-specific recombination achieved by introducing the Cre-LoxP system from the PI bacteriophage into mouse embryo (ES) cells. The basis for the invention describes an experimental system for the investigation of a novel endogenous recombination process in mammalian cells. This system involves a unique mutant clone (Sp5) of Chinese hamster V79 cells exhibiting a displaced duplication in the hprt locus (Zhang and Jenssen, 1992). This locus has the advantage that both forward and reverse mutations can be isolated and analyzed by DNA sequencing. The Sp5 clone investigated here was originally isolated from a set of mutants obtained during studies of spontaneously occurring mutations (Zhang and Jenssen, 1989; Zhang et al, 1992). This Sp5 clone exhibited the interesting characteristic of having a remarkably high reversion frequency (3.7 x 10 "5 ), indicating an instability of the hprt locus alone (Zhang and Jenssen, 1992). This high reversion frequency could sometimes be elevated even further by treatment with chemical and physical agents (Zhang and Jenssen, 1994). 4 In a previous report utilizing Sp5 cells (Zhang and Jenssen, 1992), Southern blot analysis indicated the size of the inserted duplication to be approximately 2.1 kb, containing exon 2 and its flanking intron sequences and located in a region of unknown sequence identity between the two Eco RI sites of intron 1. The displaced form of duplication found for the Sp5 mutant was unique and different from other duplications mutants isolated from the same parent cell line (V79) which were further characterized by Southern blot analysis (Dare et al, 1996). The Invention The invention is defined in the attached claims. Description of the invention The information for this invention is based on experimental data on a specific clone of the V79 cell line. This cell line originates from lung tissue of a male Chinese hamster. The clone named Sp5 by us was isolated as a mutation of V79 cells (1-5) in the gene for hypoxanthine-guanine phosphoribosyl transferase (hprt) (6). The Sp5 clone has now been found to be unique in its genetic constitution concerning the hprt-gene and has now been found to have the following specific characteristics: - A sequence of 2.1 kb surrounding exon 2 in the hprt-ge has by an endogenous recombination mechanism been duplicated, modified in its ends and inserted at a displaced position within an until now unknown sequence between to Eco RI restriction sites in intron 1 of the same gene. (Figure 1). - The Eco RI restriction fragment sequence of intron 1 of the hprt gene of V79 cells was analyzed and found to included totally 956 bp sequence and a direct repeat of 11 bases comprising a 5 base pair repeat unit and one base spacer (TCTTT 5 T TCTTT) beginning at a position 197 bases downstream the 5' Eco RI site (See Example 1 and figure 2). - The insert sequence of 2.1 kb is characterized by direct repeat sequences in its ends, each repeat consisting of 5 plus 5 or 6 base pairs separated by a spacer of one base pair. (See Example 2). - Revertance of Sp5 cells occur at frequencies of approximately 1 x 10"5 per cell generation. Revertant clones are characterized by exact removal of the inserted sequence in 16 out of 16 clones investigated. (See Example 3). The sequence in intron 1 , which is restored by site specific recombination is characterized by a direct repeat sequence of 10 bases and one base spacer (TCTTT T TCTTT). - This endogenous type of recombination in Sp5 is unique since its characteristics are not consistent with other known recombination mechanisms in somatic mammalian cells. An overview of the characteristics of similar but not identical recombination mechanisms described in the literature is listed in Table 1. The data compiled here gives the possibility to create or to remove duplicated, inversed or other types of rearranged sequences in a controlled manner. Such recombination process is a new recombination mechanism here found to be present in Sp5 cells, probably involving cellular proteins, which executes intragenic recombination characterized by the necessity of specific direct repeats as recognition sequences for executing the process. Thus, the specificity and unique property of this new process is the basis for an invention of a tool to in a controlled way remove or in any other way rearrange above described unwanted sequences in mammalian cells when such sequences are left after using different now available tools for genetic engineering of mammalian cells. A comparison between the Sp5 recombination system according to our invention and other site-specific recombination systems is found in Table 1. 6 Table 1. Comparison of known site-specific recombination processes. From Table 1 it is thus clear that the claimed Sp5 recombination process is unique in comparison to other known site-specific recombination mechanisms and until now, except for V(D)J recombination, the only endogenous recombination process yet described to occur in mammalian cells. V(D)J recombination has not been reported to be useful for genetic engineering in mammalian cells and has the disadvantage to execute recombination by junctional flexibility. Dead end product means that the recombination mechanism will not leave the recognition site sequence (RSS), thus making the recombination process irreversible. The reason why the Cre-LoxP systemt in transgenic mice is unfavoured to the Sp5 mechanism is because it requires insertion of recognitions sites from bacteria and it also requires expression of non-mammalian Cre protein in the tissue that is subjected for recombination. Transposons could be used for gene targeting but with unspecific integration, which is a major disadvantage to the Sp5 recombination system. V(D)J recombination is exclusively executed in premature lymphocytes and some lymphoblastic cell lines, with the RAGl and RAG2 proteins which have not been reported to be present in mammalian non- lymphatic cells. Figure 1. Illustration of the genetic constitution of the Sp5 clone. In Figure 1 the specific genetic constitution of the Sp5 clone is illustrated as compared to the wild type hprt gene of Chinese hamster V79 cells as described by Rossiter et al, 1991. Figure 2. The sequence of the region for insert of a duplicated fragment in the Sp5 clone. The sequence of the wild-type DNA fragment between two Eco RI sites in intron 1 is shown in Figure 2 revealing a direct repeat at the position of insert of the duplicated region characterizing the Sp5 mutant clone. The sequence identity Eco RI fragment was revealed according to Example 1. The repeat sequence at the insert position in this 7 example is indicated by brackets. The position of insert of the duplicate of the SP5 clone is indicated by an arrow. Figure 3. The sequence of the repeats flanking the duplicated sequence of the Sp5 clone. Figure 3 show parts of the results of the DNA-sequence analysis of the duplicated fragment, e.g., the inserted sequence that expanded the Eco RI fragment from 956 bp of wild type cells to 2.1 kb in the Sp5 mutant. The procedure and method for the analysis of the inserted sequence is indicated in Example 2. Only the 5' and 3' ends of the results of the sequencing of the inserted fragment is indicated in Figure 3. The results according to Example 2 showed that both the 5' and 3' ends the inserted fragment of the Sp5 clone consisted of repeat sequences characterized by repeat unit of 5 plus 5 or 6 base pairs separated by one base spacer. Figure 4. Schematic illustration of steps involved in the site-specific recombination process restoring the Sp5 clone to wild type. In Figures 4a-c, the recombination steps involved in the precise restoration of the wild- type sequence of the hprt gene of Chinese hamster V79 cells are illustrated schematically. The process suggested is based on the results obtained in Example 1-3. The first step (Figure 4a) may involve binding of protein(s) to the repeat sequences terminating the duplicate followed by formation of a loop structure (Figure 4b) and release of an episome. The restored wild-type hprt sequence (Figure 4c, underlined) of the Sp5 revertants is indicated here as the DNA sequence flanking the insert position (indicated with an arrow). The results of the analysis of the Sp5 mutant, schematically illustrated in Figures 4a-c, indicate a mechanisms involving recognition by proteins according to the knowledge of similar sequences in lower organisms. The full wild type sequence of the EcoRI fragment (956 bp) of intron 1 is shown in Figure 1. The repeats involved in the position of the insert are indicated underlined. 8 Example 1. Material and methods for identifying the exact location of the duplicated fragment of the Sp5 clone: To resolve the molecular structure of the Sp5 clone, the cells were cultured in Hank's Minimal Essential Medium (Gibco) supplemented with 8% fetal calf serum (Gibco) and 5IU/5 μg Penicillin/Streptomycin (HMEM) and containing 5μg 6-thioguanine/ml as described previously (Zhang and Jenssen, 1992). The inserted duplicated region was located earlier to be within the EcoRI-fragment of intron 1 (Zhang and Jenssen, 1992). Before sequencing the inserted duplicated region (2,lkb) of Sp5, the wild-type DNA fragment between two Eco RI sites in intron 1 (see Figure 1) had to be sequenced. This DNA fragment, kindly provided by Dr. B. Rossiter, was cloned into Ml 3 vector mpl9 by standard procedures and sequenced using the dideoxy chain termination procedure (Sanger et al, 1977). The sequence obtained is shown in Figure 2. Example 2. The method for sequencing the inserted duplication To be able to reveal the sequence of the inserted duplicated fragment of the Sp5 clone, a DNA lysate was prepared from cells cultured according to Example 1. Thus, 20-30 xlO^ Sp5 cells were centrifuged 150xg for 10 min , washed two times in 15 ml of PBS and then resuspended in 1 ml PBS. The cells were subsequently centrifuged 750xg for 2 min, the supernatant removed, and 1 ml non-buffer solution [6ml 10 mg protein K/ml, 894ml dH 2 O, 100 ml lOx non buffer (50 mM KCl, 10 mM TrisCl (pH8.3) 2.5 mM MgCl2, 0.1 mg gelatin/ml, 0,45% nonidet P-40, 0.45% Tween 20)] was added. This mixture was incubated for lh at 50°-60°C and thereafter for 10 min at 95°C and was then stored at - 20°C. Using the Sp5 lysate, PCR was performed as described by Saiki et al. (1988) was performed on genomic hprt DNA. A PCR product from the 5' region of the insert of Sp5 was obtained using the primers RP5b (5' biotin-TTTTTCCCTTTTCTTCCTCCAGC 3') and Zee4w (5' TTGTGGAAAAGGTTTAGGAGCTGTA 3') using optimal conditions 9 for PCR, e.g., 94°C for 1 min; (94°C for 15 s; 58°C for 1 min; 68°C for 1 min)x40; 72°C for 8 min.; stored at 4°C. The primers used to obtain a PCR product of the 3' region of the Sp5 insert were RP4(b) (5' biotin-GGGTGGGGATCTGGTTCATGTCA 3') and Zee3 (5' GATGATGAACCAGGCTATGAC 3') and the optimal conditions were 94°C for 1 min; (94°C for 15 s; 55°C for 1 min; 68°C for 3 min)x40; 72°C for 8 min.; stored at 4°C. PCR products were electrophoresed on a 1% NuSieve gel (300 ml TBExl with 15ml EtBr) at 80V for 4h. The band was visualized using a UV -table and cut out with a razor. 20ml Dynabeads (Dynabeads M280 Streptavidin, Dynal AS) were pre-washed three times in 100ml TWS (100 mM NaCl, 10 mM TrisCl (pH=7.6), 1 mM EDTA, 0.17% Triton X- 100) and dissolved in 125ml dH2θ and 125ml SSPEx20. The isolated band was melted at 65°C for 10 min and then mixed with the Dynabead suspension. This mixture was heated at 65°C for 5 min before incubation at 37°C for at least 30 min. 300ml TWS (65°C) was added to the mixture, which was then immediately centrifuged 12,000xg for 1 min and placed in a magnetic rack. The supernatant was discarded and the pellet washed three times in 200ml TWS (at 65°C). All TWS was discarded after the final washing. 20ml lOOmM NaOH was added and the DNA denatured for 5 min. This mixture was centrifuged 12,000xg for 1 min. The resulting supernatant was collected in the tubes in the magnetic rack and transferred to new tubes and neutralized with 12.5ml 160mM HCl and 2ml 1M TrisCl (pH 8.3). The pellets were dissolved in 30ml d^O and 4.5ml 2M sodium acetate (pH 8.3). DNA sequences were determined by the dideoxy chain termination procedure (Sanger et al, 1977) using the Taq Dye Deoxy Terminator Cycle Sequencing Kit according to the supplier's protocol (Prism Ready Reaction, Applied Biosy stems) and an ABI 373 DNA sequencer. All sequences were determined in both directions at least twice. 10 The result of the sequencing of the between EcoRI sites of intron inserted region involving exon 2 showed repat sequences at both ends which are indicated in the figure 3. Example 3. Analysis of the mechanism of reversion of the Sp5 clone The region in intron 1 of the revertants, which contains the duplication in the Sp5 clone, was sequenced. Lysate of 16 isolated revertant colonies was obtained according to procedure in Example 2. A PCR product of 241 bp within the 956 bp fragment in intron 1 was obtained using the two primers RP4(b) and RP5 (See Example 2). Optimal conditions for the PCR reaction were found to be 94°C for 1 min; (94°C for 1 min; 60°C for 1 min; 72°C for 1 min)x40; 72°C for 8 min; stored at 4°C. The sequencing of the amplified DNA was performed according to Example 2 using primers RP4(b) and RP5. All 16 revertants showed exact restoration of the wild type sequence. These results implicate that a site-specific recombination process had executed the loss of the inserted fragment and that such a process is the dominating process to execute revertance of the Sp5 clone since no revertant showed sequence variation in the insert region. Applications of invention 1) Any DNA sequence of interest can be flanked by the recognition signals found here (see figure 3) and integrated into a mammalian genome by gene targeting. After integration of such a DNA fragment, the sequence between the recognition signals can be excised in a controlled way. Furthermore, any DNA sequence can be introduced into the genome at a specific site and by that provide a more specific way of integrating wanted sequences. 2) When vectors are constructed for genes to be expressed in mammalian cells such contracts also includes genes that express resistance to drugs, e.g., neomycin and zeocin. The problem using these resistance genes are several, i.e., the number of 11 cotransfected vectors are limited by the number of resistance genes and overexpression of foreign resistance proteins in the mammalian cell could sometimes be unwanted. Therefore by using a vector with a resistance gene flanked by the recognition signal, these resistance genes may be lost by an endogenous mechanism when integrated into the mammalian genome. Thus, with the invention described here it will be possible to use an unlimited amount of expression vectors without having overexpression of proteins giving drug resistance. - The expression level of a gene introduced into a genome is dependent on the position of insert into the genome. Also, insertion a foreign DNA might inactivate genes at the position of insert. When taken gene therapy in consideration these facts might cause problems, especially for the reproducibility of experiments. By having a vector that is introduced at a selected site of the genome, it is possible to control the expression level of the integrated gene without disturbing expression of other genes. Publications 1 Dare, E., Zhang, L-H. and Jenssen, D. (1996) Characterization of insertion mutants involving partial exon duplications in the hprt gene of Chinese hamster V79 cells, Somatic Cells and Molecular Genetics 22:3, 201-210. 2 Gellert, M. (1992). Molecular analysis of V(D)J recombination. Annu. Rev. Genet. 22:425-446. 3 Kilby, N., Snaith, M. and Murray, J. (1993). Site-specific recombinases: tools for genomic engineering. Trends Genet. 9:413-421. 4 McBlane, J.F., van Gent, D.C., Ramsden, D.A., Romeo, C, Cuomo, C.A., Gellert, M. and Oettinger, M.A. (1995) Cleavage at a V(D)J recombination signal requires only RAGl and RAG2 proteins and occurs in two steps. Cell 83:387-395. 5 Ramirez-Solis, R., Pentao, L. and Bradley, A. (1995) Chromosome engeneering in mice. Nature 378:720-724. 12 Rossiter, B., Fuscoe, J., Muzny, D., Fox, M. and Caskey, T. (1991). The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen. Genomics 9:247-256. Saiki, R. K., Gelfand, D. H., Stoffel, S., Schaft, S., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich., H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with chain- termination inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467. Smith, A.J.H., De Sousa, M.A., Kwabi-Addo, B., Heppell-Parton, A., Impey, H. and Rabbitts P. (1995). A site directed chromosomal translocation in embryonic stem cells by Cre-LoxP recombination. Nat. Genet. 9:376-385. Stark, W.M., Boocock, M.R. and Sherratt, D. J. (1992) Catalysis by site-specific recombinases. Trends Genet. 8:432-439. Van Gent, D.C., Ramsden, D. A. and Gellert, M. (1996) The RAGl and RAG2 proteins establish the 12/23 rule in V(D) J recombination. Cell 85 : 107- 113. Zhang, L.-H. and D. Jenssen (1989) Isolation and characterization of spontaneously occurring mutations at the HGPRT locus in V79 Chinese hamster cells, Mutation Res. , 212, 253-262. Zhang, L.-H. and Jenssen, D. (1992a). Reversion of the hprt mutant clone SP5 by intrachromosomal recombination. Carcinogenesis 13:609-615. Zhang, L.-H. and Jenssen, D. (1994). Studies on intrachromosomal recombination in SP5 /V79 Chinese hamster cells upon exposure to different agents related to carcinogenesis. Carcinogenesis 15:2303-2310. Zhang, L.-H., Vrieling, H., van Zeeland, A. A. and Jenssen, D. (1992b). Spectrum of spontaneously occuring mutations in the hprt gene of V79 Chinese hamster cells. J. Mol Biol. 223:627-635. w o 0 *0- Table 1. Criteria showing that Sp5 recombination is unique in comparison to other known site-specific recombination mechanisms Cre-/oxP V(D)J Sp5 Property recombination Transposons recombination recombination Recognition site -13 bp inverted Inverted repeats Heptamer, 12/23 8-9 bp direct sequence (RSS) repeats, -8 bp spacer, nonamer repeat, 1 bp spacer spacer Joining Precise Precise Junctional flexibility Precise mechanism Sequence product Restored site Restored site or Dead end product Dead end product Dead end product Cleavage Core region Flanked at side Flanked at side Flanked at "0 o of RSS of RSS side of RSS H «5 M o © o <Λ

Claims: 14 CLAIMS 1. DNA sequence of 956 base pairs as given in Figure 2, sequence list 1. 2. DNA sequence including two repetitive sequences consisting of ATTGATTAT or ATT ATT AT as repeat units separated by a spacer of one base pair at 5 ' end and of two TGTATAATA as repeat units separated by a spacer of one base pair at 3 ' end. 3. DNA sequence according to claim 1 additionally including the DNA sequence according to claim 2 in a position after 204 as indicated by J) in the TCTTT T TC TTT sequence of Figure 2. 4. DNA sequence, which is a duplication fragment of 2.1 kb of the type specific to the Sp5 clone as described in Figure 3, including a copy of exon 2 and is flanked by the DNA sequence according to claim 2. 5. A method for a site specific recombinogenic process, characterized by inserting sequence of interest comprising repeat sequences according to claim 2 in each end and removing this sequence of interest in an exact and controlled way by an endogenous process. 15 6. A method for introducing a gene of interest into mammalian cells comprising the steps of: inserting DNA sequence comprising a selective marker and a sequence that is homologous to the site of desire and also includes the sequence TCTTT T TCTTT to produce a target site integrating an additional sequence comprising a gene of interest and in both ends repeat sequences according to claim 2, integrating this into the target site executed by an endogenous process. 7. Method for removal of sequences previously introduced by any gene targeting method characterized by introducing a DNA sequence by endogenous recombination, such DNA sequence includes the repeat sequences in claim 2 removing any unwanted sequence executed by endogenous process that recognize these repeat sequences to be excised. 8. Use of a direct repeat sequence of 10 bases and one base spacer, 5 '-TCTTT T TCTTT - 3' for transgenic work. 9. Use of 5'-TCTTT T TCTTT - 3' for recombination in mammalian cells. 10. Use of 5 '-TCTTT T TCTTT - 3 ' as target site. 11. Use of sequences according to any of claims 1 , 2 or 3 as a tool of site specific recombination.

Inventors: HELLEDAY THOMAS, JENSEN DAG, HELLEDAY, THOMAS, JENSEN, DAG
Applicants: GENOTOX TESTING AND CONSULTING, HELLEDAY THOMAS, JENSEN DAG, GENOTOX TESTING AND CONSULTING HB, HELLEDAY, THOMAS, JENSEN, DAG
Application year: 1999
Main classification: C12N15
IPC codes: C12N9, C12N15