An improved system of somatic cell molecular genetics for analyzing the requirements of Ig synthesis and function.
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)
Oancea
A E
AE
Department of Immunology, University of Toronto, Canada.
Shulman
M J
MJ
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D006825:Hybridomas / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000235:genetics; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D008967:Molecular Biology / Q000379:methods; D011995:Recombination, Genetic / Q000235:genetics
D005819:Genetic Markers; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007148:Immunoglobulin mu-Chains