Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
AssigneeInstitut Pasteur Universite Paris-VI
InventorsBernard Dujon, Andre Choulika, Laurence Colleaux, Cecile Fairhead, Arnaud Perrin, Anne Plessis, Agnes Thierry
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Abstract
An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.
Claims29
An isolated DNA encoding the enzyme I-SceI, wherein the DNA has the nucleotide squence (SEQ ID NO.1):
- M H ATG AAA AAC ATC AAA AAA AAC CAG GTA ATG 2670 M K N I K K N Q V M 12 2671 AAC CTC GGT CCG AAC TCT AAA CTG CTG AAA 13 N L G P N S K L L K GAA TAC AAA TCC CAG CTG ATC GAA CTG AAC 2730 E Y K S Q L I E L N 32 2731 ATC GAA CAG TTC GAA GCA GGT ATC GGT CTG 33 I E Q F E A G I G L ATC CTG GGT GAT GCT TAC ATC CGT TCT CGT 2790 I L G D A Y I R S R 52 2791 GAT GAA GGT AAA ACC TAC TGT ATG CAG TTC 53 D E G K T Y C M Q F GAG TGG AAA AAC AAA GCA TAC ATG GAC CAC 2850 E W K N K A Y M D H 72 2851 GTA TGT CTG CTG TAC GAT CAG TGG GTA CTG 73 V C L L Y D Q W V L TCC CCG CCG CAC AAA AAA GAA CGT GTT AAC 2910 S P P H K K E R V N 92 2911 CAC CTG GGT AAC CTG GTA ATC ACC TGG GGC 93 H L G N L V I T W G GCC CAG ACT TTC AAA CAC CAA GCT TTC AAC 2970 A Q T F K H Q A F N 112 2971 AAA CTG GCT AAC CTG TTC ATC GTT AAC AAC 113 K L A N L F I V N N AAA AAA ACC ATC CCG AAC AAC CTG GTT GAA 3030 K K T I P N N L V E 132 3031 AAC TAC CTG ACC CCG ATG TCT CTG GCA TAC 133 N Y L T P M S L A Y TGG TTC ATG GAT GAT GGT GGT AAA TGG GAT 3090 W F M D D G G K W D 152 3091 TAC AAC AAA AAC TCT ACC AAC AAA TCG ATC 153 Y N K N S T N K S I GTA CTG AAC ACC CAG TCT TTC ACT TTC GAA 3150 V L N T Q S F T F E 172 3151 GAA GTA GAA TAC CTG GTT AAG GGT CTG CGT 173 E V E Y L V K G L R AAC AAA TTC CAA CTG AAC TGT TAC GTA AAA 3210 N K F Q L N C Y V K 192 3211 ATC AAC AAA AAC AAA CCG ATC ATC TAC ATC 193 I N K N K P I I Y I GAT TCT ATG TCT TAC CTG ATC TTC TAC AAC 3270 D S M S Y L I F Y N 212 3271 CTG ATC AAA CCG TAC CTG ATC CCG CAG ATG 213 L I K P Y L I P Q M ATG TAC AAA CTG CCG AAC ACT ATC TCC TCC 3330 M Y K L P N T I S S 232 3331 GAA ACT TTC CTG AAA TAA 233 E T F L K *
A method for in vivo site directed genetic recombination in an organism using enzyme I-SceI, comprising the steps of:
- (a) providing an expression vector comprising a synthetic gene encoding I-SceI endonuclease;
- (b) providing a plasmid comprising a I-SceI restriction site next to or within a gene of interest carried on said plasmid;
- (c) co-transforming the cells of said organism with said expression vector of step (a) and said plasmid of step (b);
- and
- cleaving said I-SceI restriction site, whereby said cleavage promotes the insertion of said gene of interest into a chromosome of said organism at a specific site by homologous recombination.
A method for in vivo site directed genetic recombination in an organism using enzyme I-SceI, comprising the steps of:
- (a) providing a transgenic cell in which at least one I-SceI recognition site is inserted at a unique location in a chromosome;
- (b) providing an expression vector comprising a synthetic gene encoding I-SceI endonuclease;
- (c) providing a plasmid comprising a gene of interest and a DNA sequence homologous to the sequence of the chromosomal DNA, allowing homologous recombination into which the I-SceI site is inserted;
- (d) co-transforming said transgenic cell with said expression vector of step (b) and said plasmid of step (c);
- and
- cleaving said I-SceI restriction site, whereby said cleavage promotes the insertion of said gene of interest into said chromosome of said organism at a specific site by homologous recombination.
Dependencies are inferred from the claim text.