A hit-and-run system for introducing mutations into the Ig H chain locus of hybridoma cells by homologous recombination.
The traditional method of site-specific mutagenesis is to introduce predetermined mutations into an expression vector, which is then transferred to cells so that the relevant gene product and the effects of the mutations can be measured. A problem with this approach is that the expression of the transferred genes varies from transformant to transformant, presumably because the number of vector copies and the site of chromosomal integration vary among transformants. While it should be possible to avoid this variability by mutagenizing the chromosomal gene itself, the labor involved in introducing predetermined mutations by homologous recombination with a mutagenized vector is usually so intense that this has not been the favored method. We describe here a system for introducing mutations into the IgH locus of hybridoma cells. This system greatly reduces the labor that would usually be required to identify and recover the rare recombinants. This is a two-step, so-called "hit-and-run," method, whereby mutations are first introduced into the chromosomal locus by targeted vector integration, after which the vector is excised so as to leave the mutation in the chromosomal target. The first step employs an enhancer trap vector bearing an enhancerless gpt gene; using this vector the frequency of randomly inserted transformants which grow in mycophenolic acid containing selective medium is so low that approximately 25% of the selected transformants have integrated the vector into the IgH locus by homologous recombination. Properly targeted transformants can then be used to derive secondary recombinants that have excised the vector and thus become gpt-. This second step which involves selection of gpt- cells by their resistance to 6-thioxanthine is also efficient, in that approximately 75% of the treated cells have excised the gpt gene by homologous recombination. Overall the labor involved in mutagenizing the chromosomal locus is not much more than is needed to produce the traditional transformants expressing a mutagenized transferred gene.
Bautista
D
D
Department of Immunology, University of Toronto, Canada.
Shulman
M J
MJ
D001402:B-Lymphocytes / Q000378:metabolism*; D015139:Blotting, Southern; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006825:Hybridomas; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D066298:In Vitro Techniques; D016254:Mutagenesis, Insertional / Q000379:methods*; D011995:Recombination, Genetic
D007148:Immunoglobulin mu-Chains