Cosmid vectors for high efficiency DNA-mediated transformation and gene amplification in mammalian cells: studies with the human growth hormone gene.
We have constructed two new recombinant cosmid vectors that can be used for direct expression and amplification of genomic DNA in mammalian cells. The vectors allow cloning of DNA fragments up to 40 kb in size. Each carries two dominant selectable markers: the bacterial neo gene and the mouse DHFR gene. In the first vector, pCV001, the neo and DHFR genes are regulated by the SV40 early promoter, and in the second, pAVCV007, by the avian sarcoma virus LTR promoter. The neo gene served as a dominant marker for the selection of transformants in all mammalian cell types, and we demonstrate here that the LTR promoter significantly improved the efficiency of DNA-mediated transformation of a human cell line. We isolated the human growth hormone genes from genomic libraries prepared in these cosmid vectors and used these recombinant cosmids for direct transfections of cultured cells. Selection of transformants in increasing concentrations of methotrexate led to the outgrowth of resistant cell populations carrying amplified copies of the DHFR marker. A 40-1000-fold coamplification of the hGH genes was observed in the different transfected cell lines, along with a corresponding increase in transcription and translation activity of the hGH gene. Gene amplification could be achieved in both DHFR deficient or normal cell lines. High level expression of a cloned gene mediated by gene amplification should facilitate characterization of DNA sequences, as well as isolation of specific gene products for biochemical, functional, and pharmacological studies.
Choo
K H
KH
Filby
G
G
Greco
S
S
Lau
Y F
YF
Kan
Y W
YW
D000818:Animals; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003360:Cosmids; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics*; D006801:Humans; D051379:Mice; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014170:Transformation, Genetic
D005819:Genetic Markers; D013006:Growth Hormone; D013762:Tetrahydrofolate Dehydrogenase