Mouse λ light chain locus

• US 7,541,513 B2
• Filed: 06/21/2002
• Issued: 06/02/2009
• Est. Priority Date: 06/21/2001
• Status: Expired due to Fees

• Current Assignee: Barbraham Institute, Crescendo Biologics Limited
• Original Assignee: Translocus Limited
• Inventors: Bruggeman, Marianne, Zou, Xiangang
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US10/481,395
• Publication Number:
  US 20050060763A1
• Time in Patent Office: 2,538 Days
• Field of Search: 800/1, 800/3, 800/20, 800/22, 800/21
• US Class Current: 800/22
• CPC Class Codes: A01K 2217/058 due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/06 from serum
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

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.

Claims

1. A method of making 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 method comprises:

(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. A method of making a double knock-out mouse that has both the lambda light chain and the kappa light chain loci deleted, wherein the double knock-out mouse is produced by

(I) breeding (1) a knock-out mouse having its kappa light chain locus deleted with (2) a knock-out mouse having its lambda light chain locus C2-C4-C3-C1 deleted by:

(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); and

(II) obtaining a progeny double knock-out mouse that has both the lambda light chain and the kappa light chain loci deleted by the breeding of (I).