Expression of a human O6-alkylguanine-DNA-alkyltransferase cDNA in human cells and transgenic mice.
A truncated human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA was ligated into an expression vector under the control of the mouse metallothionein-1 gene promotor and upstream of part of the human growth hormone gene to provide splice and polyadenylation signals. Transfection of this construct into human cells resulted in very high levels of ATase expression (more than 300 fmoles/mg protein versus less than 2 fm/mg protein in parent vector transfected control cells). Microinjection of a 4.2 kb fragment of this vector into B6D2F2 mouse embryos and implantation of survivors into pseudopregnant females has so far generated 35 offspring. Southern analysis of tail tip DNA has shown that 11 of the offspring are transgenic for the human ATase gene, between 1 and at least 30 copies of the gene being detected. Human ATase transcripts were detected in total RNA extracted from liver obtained from two male transgenic mice by partial hepatectomy. Cell free extracts of liver samples from five transgenic mice showed up to 4 times higher ATase levels than control livers.
Fan
C Y
CY
Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
Potter
P M
PM
Rafferty
J
J
Watson
A J
AJ
Cawkwell
L
L
Searle
P F
PF
O'Connor
P J
PJ
Margison
G P
GP
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006498:Hepatectomy; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014162:Transfection
D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase