Abstract

Alpha-lactalbumin (alpha-lac) is a component of the enzyme complex lactose synthetase which plays a key role in the regulation of lactose synthesis and milk water secretion. Ribozyme mediated manipulation of alpha-lac expression should enable more precise elucidation of its physiological significance and may lead to the development of transgenic animals. Five ribozymes and controls targeting different regions of the alpha-sac gene have been synthesised and cloned. In vitro ribozyme analysis involved the incubation of in vitro transcribed alpha- lac and ribozyme RNA. The rate of ribozyme cleavage in vitro was influenced by temperature, MgC1 2 concentration, length of antisense flanking sequences and region of the alpha-lac gene targeted. In vivo ribozyme analysis was carried out in the T7/vaccinia expression system using C127I mouse mammary cells. This system utilises the powerful bacteriophage T7 RNA polymerase which is expressed when cells are infected with a recombinant vaccina virus. Gene constructs, cloned into vaccina plasmids downstream from a T7 promoter are transfected into the cells and are transcribed and translated at very high levels. Using this system we have identified that ribozymes that targeted exposed stem loops and the 3' end of the gene down regulated the level of alpha-lac RNA to 10-20% of the controls. Similar reductions in expression of alpha-lac protein were observed for these key ribozymes, but in addition a full length antisense was equally as effective. The expression of these ribozymes in transgenic mice will enable evaluation of their effectiveness to influence lactose synthesis and milk water secretion.

J, Lee, ND Grace, and S Martell

Proceedings of the New Zealand Society of Animal Production, Volume 51, , 173-178, 1991