Expression of a bovine kappa-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette.
Transgenic mice were produced by microinjection of a DNA construct composed of the bovine kappa-casein (kappa-CN) cDNA under the control of the goat beta-CN 5' promoter elements and 3' flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine kappa-CN RNA to the mammary gland and secretion of bovine kappa-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine kappa-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69%, and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine kappa-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine kappa-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did kappa-CN from bovine milk. A high degree of variation in the production of bovine kappa-CN within each of the transgenic lines was observed.
Gutiérrez
A
A
Department of Animal Science, University of California, Davis 95616-8521, USA.
Meade
H M
HM
Ditullio
P
P
Pollock
D
D
Harvey
M
M
Jiménez-Flores
R
R
Anderson
G B
GB
Murray
J D
JD
Medrano
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000096:biosynthesis* / Q000235:genetics; D002417:Cattle; D004247:DNA / Q000008:administration & dosage; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006041:Goats; D007774:Lactation / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D008894:Milk Proteins / Q000032:analysis / Q000096:biosynthesis; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping
D002364:Caseins; D008894:Milk Proteins; D004247:DNA