Gene targeting in embryonic stem cells and the manipulation of milk composition

• Main Author: Kumar, Satish
• Published: University of Edinburgh 1994
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.653581

Mice carrying mutated β-casein genes were created through gene targeting in embryonic stem cells using a replacement-type vector. β-casein was absent from the milk of homozygous mutant mice, and no mRNA was detectable from the mutant allele. The mutation was compatible with viability, fertility and lactation. The total protein concentration of β-casein-deficient milk was reduced, although the loss of β-casein was partially compensated by enhanced synthesis and/or secretion of other milk proteins. The pre-weaning growth of pups feeding β-casein deficient milk appeared to be slower, probably reflecting the reduction in the total milk protein concentration. The reduction (in heterozygotes) or absence (in homozygotes) of β-casein in milk resulted in smaller casein micelles, probably due to increases in the ratios of κ-casein to calcium-sensitive caseins. These results show that β-casein is dispensable in lactation. Targeting of subtle mutations is likely to be important for many purposes, including the manipulation of milk structure. The 'hit and run' method for targeting the subtle mutations would be facilitated by positive-negative selection during the first (targeted insertion) step. To explore the feasibility of negative selection in conjunction with insertion-type gene targeting vectors, the effects of placing terminal heterologies on a hypoxanthine phosphoribosyltransferase (HPRT) insertions vector were determined. The targeting was generally accurate but the targeting efficiency was reduced by terminal heterologies.