An improved system of somatic cell molecular genetics for analyzing the requirements of Ig synthesis and function

Abstract

A general method of relating molecular function and structure is to examine the biological and chemical effects of defined mutations. In many cases, particularly those concerned with the rate or efficiency of gene expression, it is important to assess mutations in the normal chromosomal context. There are two methods of obtaining such mutants: (i) site-directed mutagenesis of the chromosomal locus, using homologous recombination to target defined mutations to the gene of interest, and (ii) phenotypic selection of mutant organisms. For most mammalian genes the rarity of targeted recombinants and phenotypically evident mutants impede the use of either of these approaches. However, various genetic and biochemical features render the Ig heavy chain locus in B cell lines amenable to both gene targeting and phenotypic selection of mutants. We describe here a replacement-type vector in which the selectable marker is an enhancerless gpt gene which is particularly suitable for targeting the IgH locus. Deletion of the enhancer greatly decreased the frequency of gpt+ random transformants while still allowing properly targeted transformants to be gpt+, such that transformants with the predicted recombinant structure comprised 25% of the gpt+ population. Thus, the labor involved in mutagenizing the chromosomal locus using this method is comparable to the usual method of isolating randomly inserted transformants, but offers the important advantages that the copy number and integration site are the same in independent transformants. In the hybridoma cell lines which we have tested, the consistent copy number and integration site are sufficient to yield a uniform level of recombinant gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Complex regulation of somatic hypermutation by cis-acting sequences in the endogenous IgH gene in hybridoma cells

“…All recombinants were derived by homologous recombination from a derivative of Sp6, igm692 (16). The WT recombinant has the WT complement of intronic regulatory elements (17). In E, only the core E enhancer is present in the J-C intron (18).…”

“…The WT recombinant was generated by inserting the guanyl phophorybosyl transferase (gpt) between the C region and C␦ region ( Fig. 1 A) (17), resulting in insulation from the influence of the 3Ј locus control region on the expression of the heavy chain (20). In WT, all of the cells still expressed WT levels of IgM (14).…”

Expression of the (Recombinant) Endogenous Immunoglobulin Heavy-Chain Locus Requires the Intronic Matrix Attachment Regions

“…2B, a transfer vector bearing a normal C region and the gpt gene can recombine with the mutant gene of igm692, such that the recombinant produces normal and is mycophenolic acid resistant (MHX R ). We have shown that insertion of the gpt gene in this manner does not alter the level of expression, i.e., that such recombinants transcribe the gene at the same rate as the igm692 and Sp603 parental cell lines (30,31). By using vectors with altered 5Ј homology regions, gene targeting can be applied to delete segments of the J H -C intron.…”

“…2 (30). This system is based on the wild-type hybridoma Sp603, which has a single copy of the gene and produces IgM() specific for the hapten trinitrophenol.…”

“…In contrast to the foregoing analyses, we have developed a simple system for modifying the gene in the IgH locus of hybridoma cells in which insertion of the vector-borne selectable marker does not alter normal expression (30). As reported here, we have used this system to test the effects on expression of deleting segments of the J H -C intron.…”