Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a newin vivo amino acid biosynthesis pathway and improved wool growth

Abstract

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesise cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes of Salmonella typhimurium, cysE, cysM and cysK, and linked cysEM, cysME and cysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and -cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and -cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cells in vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.

The Role of Biotechnology in Improvement of Livestock

“…The approach was successfully demonstrated with two different bacterial genes in transgenic mice, while this approach was unsuccessful in sheep due to low level of unsustainable biosynthetic enzyme expression (Ward et al 1994;Bawden et al 1995;Ward 2000). Cysteine is a wool growth regulating element in livestock rear for wool or fiber production.…”

Biology of the wool follicle: an excursion into a unique tissue interaction system waiting to be re‐discovered

“…The strategy would require efficient expression of the enzymes using promoters that establish function in the epithelial lining of the rumen wall. Expression of the enzymes in vivo (104) and in cell lines (105) has been reported but has not been further investigated.…”

Altering nutrient utilization in animals through transgenesis

“…On the other hand, the pathway appears to be less compatible with the existing intermediary metabolism of sheep. A summary of the results obtained in our own laboratory and that of Rogers and colleagues (Sivaprasad et al 1992;Bawden et al 1995) is provided by Ward et al (1998). Brie¯y, these show that twenty-eight transgenic sheep with the genes encoding cysteine biosynthesis have been produced.…”

“…This ®eld is progressing rapidly as structure ±function predictions of proteins become more sophisticated and promises to be an effective way of reducing the amount of DNA required to encode metabolic pathways. A closely related example in plants consists of a three-enzyme complex designed to catalyse a portion of the mannityl opine biosynthetic pathway complex (von Bawden et al 1995). The gene was constructed by fusing the relevant coding sequences together and placing a single strong plant promoter at the 5 H end of the complex.…”