Isolation of cDNA clones specific for collagen IV and laminin from mouse teratocarcinoma cells.

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Abstract
The synthesis of the protein laminin and collagen IV is stimulated .apprxeq. 20-fold in F9 mouse teratocarcinoma stem cells after treatment of the cells with retinoic acid and N6, O2''-dibutyryl-cAMP (Bt2cAMP). A c[complementary]DNA library from F9 cells treated with retinoic acid, Bt2cAMP, and theophylline (F9-R + DBC cells) was constructed to isolate cDNA coding for collagen IV or laminin. The recombinant plasmids were screened by differential colony hybridization to cDNA synthesized from poly(A)+ RNA isolated from F9 stem and F9-R + DBC cells. Differentially hybridizing plasmids were then used as probes to hybridize to RNA transfer blots to determine the size of their specific mRNA. Only plasmids containing cDNA sequences specific for high-MW mRNA were further analyzed. Studies by hybridization-selection, in vitro translation and immunoprecipitation showed that a plasmid clone, pc15, contains cDNA homologous to collagen IV (.alpha.2) mRNA and another plasmid clone, pc156, contains cDNA homologous to laminin B mRNA. By RNA blot analyses, the size of mRNA coding for collagen IV (.alpha.2) is 7.6 kilobases; the size of mRNA for laminin is 6.8 kilobases. Using the technique of RNA blot hybridization, the time course of the increase in mRNA coding for collagen IV (.alpha.2) and laminin B was studied in F9 cells after retinoic acid and Bt2cAMP treatment. Both collagen IV (.alpha.2) and laminin B mRNA are present in F9 stem cells. Collagen IV (.alpha.2) mRNA and laminin B mRNA levels increase slightly at .apprx. 12 h after retinoic acid and Bt2cAMP addition, with a dramatic increase between 12 and 24 h after drug treatment.