Transformation ofmousefibroblasts tomethotrexate resistance by arecombinant plasmid expressing aprokaryotic dihydrofolate

Arecombinant plasmid hasbeen constructed for theexpression ofinserted DNAsequences coding for polypeptide chains using thesimian virus 40early promoter andsplicing and polyadenylylation signals from therabbit f-globin gene. Thecod- ingregions fortwoprokaryotic methotrexate-resistant dihydro- folate reductases wereintroduced into theexpression vector. Whenmouse fibroblasts wereexposed tothese recombinant plas- mids, itwaspossible toselect methotrexate-resistant clones that hadintegrated theplasmids andproduced achimeric RNAcoding fortheprokaryotic enzyme. Thedevelopment ofsystems both invivo andinvitro inwhich genes arefaithfully expressed isoneofthemajor aims ofcurrent research ineukaryotic molecular biology. While methods have been described for thetransfer ofDNAinto cells inculture (1, 2), theproportion ofcells that take uptheforeign DNAisso small that aselection procedure isrequired inorder toobtain sufficient material toanalyze expression oftheforeign DNA sequences. Muchofthepioneer work inthis direction hasused thethymidine kinase (TK) genefromherpes simplex virus as aselective marker, butthis could only beused with recipient TK-cell lines (3-5). Adominant selective marker that could beused with anytype ofcell would represent aconsiderable advance because mostofthecell lines into which itwould be interesting totransfer genes (e.g., steroid hormone-responsive chicken genes into ahormone-sensitive cell line) arenotavail-