Mouse λ light chain locus
View Patent ↗The present invention provides in a first aspect a mouse in which the λ (lambda) light chain locus has been functionally silenced. In one embodiment, the mouse λ light chain locus was functional silenced by deletion of gene segments coding for the λ light chain locus. In a further aspect, a mouse containing functionally silenced λ and κ (kappa) L chain loci was produced. The invention is useful for the production of antibodies, for example heterologous antibodies, including heavy chain only antibodies.
1. A knock-out mouse having its lambda light chain locus C2-C4-C3-C1 deleted using LoxP constructs as the targeting constructs and ubiquitous Cre-expresser mice, wherein the knock-out mouse is produced by a method comprising the steps of:
(a) introducing at least one C3-C1 targeting construct that comprises LoxP sequences to a mouse embryonic stem (ES) cell, thereby producing a mouse ES cell having silenced C1;
(b) introducing the mouse ES cell having silenced C1 to a mouse embryo in order to produce a mouse having silenced C1 by germline transmission;
(c) breeding the mouse having silenced C1 with a Cre-expresser mouse, which causes targeted deletion of C3-C1;
(d) obtaining a C3-C1 deleted mouse as a result of the breeding of (c);
(e) obtaining a mouse ES cell from the C3-C1 deleted mouse;
(f) introducing a C2-C4 targeting construct that comprises a LoxP sequence to the mouse ES cell from the C3-C1 deleted mouse, thereby producing a mouse ES cell having silenced C2;
(g) introducing the mouse ES cell having silenced C2 to a mouse embryo in order to produce a mouse having silenced C2 by germline transmission;
(h) breeding the mouse having silenced C2 with a Cre-expresser mouse, which causes targeted deletion of C2-C4; and
(i) obtaining a knock-out mouse having deleted lambda locus C2-C4-C3-C1 as a result of the breeding of (h).
2. The knock-out mouse according to claim 1 , wherein κ light chain locus is functionally silenced by targeted integration of a selectable marker gene in Cκ or targeted removal of Cκ or Jκ.
3. The knock-out mouse according to claim 1 , wherein the mouse heavy chain locus is functionally silenced by targeted integration of a selectable marker gene in the μ membrane exons or targeted deletion of JH gene segment.
4. The knock-out mouse according to claim 1 , wherein the knock-out mouse comprises one or more heavy and/or a light chain genes or loci from a human.
5. A method of producing a knock-out mouse, wherein lambda light chain locus is deleted through knock-out by targeted integration and gene deletion using LoxP constructs as the targeting constructs and ubiquitous Cre-expresser mice, wherein the method comprises the step of deleting C2-C4 loci and C3-C1 loci.
6. A method of making a knock-out mouse having its lambda light chain locus deleted, wherein the method comprises the steps of:
(a) introducing at least one C2-C4 targeting construct to a mouse embryonic stem (ES) cell, thereby producing an ES cell having silenced C2 region;
(b) introducing the ES cell having the silenced C2 region to a mouse embryo in order to produce a mouse having silenced C2 region by germline transmission;
(c) obtaining a C2 deleted mouse by breeding the mouse of step (b) with another mouse capable of causing deletion of the targeted C2-C4 region by germline transmission;
(d) obtaining ES cells from a progeny mouse of step (c);
(e) introducing a C3-C1 targeting construct to the ES cell of step (d), thereby producing an ES cell having silenced C1 region;
(f) introducing the ES cell of step e) having the silenced C1 region to a mouse embryo in order to produce a mouse having silenced C1 region by germline transmission; and
(g) obtaining a mouse having deleted lambda locus regions C2-C4-C3-C1 by breeding the mouse of step (f) with another mouse capable of causing deletion of the targeted C3-C1 region in germline transmission, wherein the lambda light chain locus is deleted by the use of LoxP constructs as the targeting constructs and by the use of a ubiquitous Cre-expresser mouse as the mouse capable of deleting a targeted region.
7. A knock-out mouse in which the lambda light chain locus is deleted through knock-out by targeted integration and gene deletion, wherein the knock-out of the lambda light chain locus is carried out by a method comprising the steps of:
(a) introducing at least one C2-C4 targeting construct to a mouse embryonic stem (ES) cell, thereby producing an ES cell having silenced C2 region;
(b) introducing the ES cell having the silenced C2 region to a mouse embryo in order to produce a mouse having silenced C2 region by germline transmission;
(c) obtaining a C2 deleted mouse by breeding the mouse of step (b) with another mouse capable of causing deletion of the targeted C2-C4 region by germline transmission;
(d) obtaining ES cells from a progeny mouse of step (c);
(e) introducing a C3-C1 targeting construct to the ES cell of step (d), thereby producing an ES cell having silenced C1 region;
(f) introducing the ES cell of step (e) having the silenced C1 region to a mouse embryo in order to produce a mouse having silenced C1 region by germline transmission; and
(g) obtaining a mouse having deleted lambda locus regions C2-C4-C3-C1 by breeding the mouse of step (f) with another mouse capable of causing deletion of the targeted C3-C1 region in germline transmission, wherein the lambda light chain locus is deleted by using LoxP constructs as the targeting constructs and using a ubiquitous Cre-expresser mouse as the mouse capable of deleting a targeted region.
8. The knock-out mouse according to claim 7 , wherein the mouse heavy chain locus is functionally silenced by targeted integration of a selectable marker gene in the μ membrane exons or targeted deletion of JH gene segment.
9. The knock-out mouse according to claim 8 , wherein the knock-out mouse comprises one or more heavy and/or a light chain genes or loci from a human.
10. The method of producing a knock-out mouse according to claim 5 , wherein the C2-C4 loci and the C3-C1 loci are deleted simultaneously.
11. The method of producing a knock-out mouse according to claim 5 , wherein the C2-C4 loci and the C3-C1 loci are deleted sequentially.
12. The knock-out mouse according to claim 2 , wherein the mouse heavy chain locus is functionally silenced by targeted integration of a selectable marker gene in the μ membrane exons or targeted deletion of JH gene segment.
13. The knock-out mouse according to claim 7 , wherein κ light chain locus is functionally silenced by targeted integration of a selectable marker gene in Cκ or targeted removal of Cκ or Jκ.
14. The knock-out mouse according to claim 13 , wherein the mouse heavy chain locus is functionally silenced by targeted integration of a selectable marker gene in the μ membrane exons or targeted deletion of JH gene segment.