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

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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: (1) site-directed mutagenesls 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 phenotyplcally 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 property 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 hybrldoma cell lines which we have tested, the consistent copy number and integration site are sufficient to yield a uniform level of recombinant gene expression. The system is therefore suitable for investigating molecular features encoded in the μ heavy chain locus which affect the rate and efficiency of immunoglobulin production. Other advantages of this system derive from the fact that the gpt gene which has been introduced into the IgH locus renders the cells sensitive to thioxanthine (TX). One application of this effect is that selection for TX resistance can be used to enrich for targeted mutations introduced by a second recombination event (balt-and-switch method). Another application concerns the possibility of identifying unanticipated expression enhancing elements in the IgH locus by selecting phenotypically for gpt defective mutants.

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