Method for modifying chromosomes
View Patent ↗The present invention relates to a method for producing a modified foreign chromosome(s) or a fragment(s) thereof, which comprises the steps of: (a) preparing a microcell comprising a foreign chromosome(s) or a fragment(s) thereof, and transferring said foreign chromosome(s) or a fragment(s) into a cell with high homologous recombination efficiency through its fusion with said microcell; (b) in said cell with high homologous recombination efficiency, inserting a targeting vector by homologous recombination into a desired site of said foreign chromosome(s) or a fragment(s) thereof, and/or a desired site of a chromosome(s) derived from said cell with high homologous recombination efficiency, thereby marking said desired site; and (c) in said cell with high homologous recombination efficiency, causing deletion and/or translocation to occur at the marked site of said foreign chromosome(s) or a fragment(s) thereof.
1. An isolated, recombinant human chromosome comprising:
(i) the SC20 chromosome fragment (Accession Number FERM BP-7583), which comprises the human chromosome #14 centromere;
(ii) two telomere sequences;
(iii) at least one recognition sequence for a site-directed recombination enzyme;
(iv) at least two fragments from different human chromosomes, wherein each fragment comprises an antibody gene, wherein one of the fragments comprises a human chromosome #14 fragment which comprises the human antibody heavy-chain gene and wherein a second chromosome fragment comprises either a human chromosome #2 fragment which comprises the human antibody light-chain kappa, or a human chromosome #22 fragment which comprises the human antibody light-chain lambda gene; and
(v) a marker gene,
wherein the at least one recognition sequence of (iii) for the site-directed recombination enzyme is located between the chromosome fragments of (iv).
2. The recombinant human chromosome of claim 1 , which is generated by chromosome recombination between the SC20 chromosome fragment and another chromosome fragment.
3. The recombinant human chromosome of claim 2 , which is generated by chromosome recombination between the chromosome fragment denoted as SC20 and a fragment of a chromosome other than the human chromosome #14.
4. The recombinant human chromosome of claim 3 , wherein the fragment of a chromosome other than the human chromosome #14 is a fragment of a human chromosome #2, which comprises a human antibody light-chain kappa gene.
5. The recombinant human chromosome of claim 3 , wherein the fragment of a chromosome other than the human chromosome #14 is a fragment of human chromosome #22, which comprises a human antibody light-chain lambda gene.
6. The recombinant human chromosome of claim 1 , wherein said recognition sequence is the loxP sequence and said site-directed recombination enzyme is the Cre recombinase.
7. The recombinant human chromosome of claim 1 , wherein said recognition sequence is the FRP sequence and said site-directed recombination enzyme is the FLP recombinase.
8. An isolated cell, comprising the recombinant human chromosome of claim 1 .
9. A method for producing a recombinant human chromosome, comprising:
(a) preparing a first isolated cell comprising a modified SC20 chromosome fragment (Accession Number FERM BP-7583), wherein the fragment comprises the human chromosome #14 centromere and the fragment is modified to contain a recognition sequence for a site-directed recombination enzyme;
(b) preparing a second, isolated cell comprising a human chromosome fragment, which comprises (i) a human antibody gene and (ii) a recognition sequence for a site-directed recombination enzyme at a desired site in said chromosome fragment;
(c) fusing said first cell with said second cell to produce a hybrid cell; and
(d) expressing a site-directed recombination in said hybrid cell,
wherein expression of said enzyme causes site-directed recombination between the modified SC20 chromosome fragment comprising the human chromosome #14 centromere, and a portion of the second chromosome fragment,
thereby producing a recombinant human chromosome.
10. The method of claim 9 , wherein said recombinant chromosome is transferred from said hybrid cell into a new cell type via microcell fusion.
11. The method of claim 10 , wherein said new cell type is a CHO cell.
12. The method of claim 9 , wherein said first cell and said second cell are chicken DT-40 cells.
13. The method of claim 9 , wherein said recognition sequence in said modified SC20 chromosome fragment of (a) and the human chromosome fragment of (b) are loxP recognition sequences and said site-directed recombination enzyme is Cre recombinase.
14. The method of claim 9 , wherein said recognition sequence in said modified SC20 chromosome fragment of (a) and the human chromosome fragment of (b) are FRP recognition sequences and said site-directed recombination enzyme is FLP recombinase.
15. The method of claim 9 , said human chromosome fragment in (b) is a fragment of either human chromosome #2, or human chromosome #22, which comprises a human antibody light chain gene.
16. The method of claim 9 , wherein said site-directed recombination is detected by the expression of a reporter gene.
17. The method of claim 16 , wherein said reporter gene is a green fluorescent protein gene.
18. The method of claim 17 , wherein said green fluorescent protein gene is obtained from the jellyfish Aequorea victoria.