Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Abstract

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre‐) B cell differentiation, we find that most Abelson murine leukemia virus (A‐MuLV)‐transformed pre‐B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A‐MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre‐B cell lines from normal mice, attempted V kappa‐to‐J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back‐to‐back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site‐specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.

Ataxia telangiectasia-mutated protein and DNA-dependent protein kinase have complementary V(D)J recombination functions

“…Genetically targeted DNA-PKcs-deficient mice showed a similar phenotype (14)(15)(16). Assays of V(D)J recombination through introduction of RAG and V(D)J recombination substrates into DNA-PKcs mutant nonlymphoid cells gave similar but not always identical results (11)(12)(13)(14)(15)(16)(17). Thus, DNA-PKcs-deficient mouse embryonic fibroblasts (MEFs) and a DNA-PKcs-deficient Chinese hamster ovary (CHO) cell line (XR-C1) had significantly decreased SJ efficiency and decreased fidelity with some SJs harboring long deletions (18)(19)(20).…”

“…The classical SCID mouse, which has a mutation in the DNAPKcs kinase domain, has a block in lymphocyte development because of inability to form V(D)J CJs in developing lymphocytes, but it still forms relatively normal SJs (11)(12)(13). Genetically targeted DNA-PKcs-deficient mice showed a similar phenotype (14)(15)(16).…”

Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation.

“…Studies of mice homozygous for the severe combined immune deficiency mutation {SCID mice), which generally lack mature B and T cells, are relevant to these issues. SCID mice have a block in B-cell development at the CD43 +, B220 + stage attributable to an impairment in the V(D)J recombination process (Hendrickson et al 1990;Lieber et al 1988;Malynn et al 1988;Blackwell et al 1989;Reichman-Fried et al 1990, 1993Biedermann et al 1991; for review, see Bosma and Carroll 1991). However, whereas introduction of a functionally assembled immunoglobulin HC gene into the SCID background generated a population of B220 +, CD43 -pre-B cells (Reichman-Fried et al 1993), introduction of HC plus LC transgenes into the homozygous SCID background has not led reproducibly to the development of significant numbers of B lymphocytes in primary or peripheral lymphoid organs (Reichman-Freid et al 1990; F. Young, R. Phillips, and F. Alt, unpubl.).…”

Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

“…DNA-PKcs mutations in mice lead to severe combined immune deficiency due to inability to form V(D)J CJs (10)(11)(12). Both point mutations in the kinase domain of DNA-PKcs (SCID mice) and elimination of DNA-PKcs expression by gene-targeted mutation abrogate CJ formation (13)(14)(15).…”