Robert Wall

Transgenic livestock: Progress and prospects for the future

Abstract The notion of directly introducing new genes or otherwise directly manipulating the genotype of an animal is conceptually straightforward and appealing because of the speed and precision with which phenotypic changes could be made. Thus, it is of little wonder that the imagination of many an animal scientist has been captivated by the success others have achieved by introducing foreign genes into mice. The private sector has embraced transgenic livestock technology resulting in the formation of two new industries. However, before transgenic farm animals become a common component of the livestock production industry, a number of formidable hurdles must be overcome. In this brief communication, the technical challenges are enumerated and possible solutions are discussed.

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 mg/ml in their milk were produced. In vitro assays demonstrated the milks ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 mg/ml of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

A rapid whole-mount staining procedure for nuclei of mammalian embryos

Abstract A rapid staining procedure for examining nuclei of mammalian embryos is described. Embryos are placed on a glass slide, counterstained with trypan blue, stained with Hoechst 33342, and embedded between the slide and coverslip in Permount. The nuclei of the embryos fluoresce brightly when examined by fluorescence microscopy immediately after staining or after extended storage. The technique has proved to be an effective tool for studying the development of cow, hamster, mouse, pig, rabbit, and sheep embryos.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15–30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln125,232-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5′-flanking region of the ovine β-lactoglobulin gene directed the secretion of Gln125,232-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

New gene transfer methods

The intentional introduction of recombinant DNA molecules into a living organism can be achieved in many ways. Viruses have been making a living by practicing gene transfer for millennia. Recently, man has gotten into the act. The paradigm employed is fairly straightforward. First, a way must be found to move genetic information across biological membrane barriers. Then, presumably, DNA repair mechanisms do the rest. The array of methods available to move DNA into the nucleus provides the flexibility necessary to transfer genes into cells as physically diverse as sperm and eggs. Some of the more promising alternative strategies such as sperm-mediated gene transfer, restriction enzyme-mediated integration, metaphase II transgenesis, and a new twist on retrovirus-mediated gene transfer will be discussed, among other methods.

Cell Donor Influences Success of Producing Cattle by Somatic Cell Nuclear Transfer

Abstract To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO2 and air and some were transferred to MEM, with 5% or 20% O2 or 0.5% or 10% serum and G418 for 2–3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.

IMPROVED YIELDS OF BOVINE BLASTOCYSTS FROM IN VITRO-PRODUCED OOCYTES. I: SELECTION OF OOCYTES AND ZYGOTES

Abstract Three experiments were conducted with 7,901 oocytes (6,152 cleaved zygotes) to identify methods to increase the proportion of oocytes aspirated from ovaries of slaughtered cows that would develop to expanded blastocysts by 9 d after exposure to spermatozoa. In the first experiment, oocytes were matured for 24 h in TCM-199, fertilized, and presumptive zygotes were co-cultured in TCM-199 and granulosa cells. Cleaved zygotes from oocytes classified as good on the basis of quality of investments and cytoplasm developed to expanded blastocysts more frequently than did zygotes from intermediate-marginal oocytes (29% vs 24%; P=0.035). In the second experiment, marginal oocytes were subdivided into those with partially expanded cumulus, coarsely granulated cytoplasm or minimal cumulus (not more than 3 layers of cumulus cells, or zona pellucida partly exposed), matured in Hams F-10, and co-cultured in Menezos B2 with buffalo rat liver (BRL) cells. Cleaved zygotes from oocytes with partially expanded cumulus and/or coarsely granulated cytoplasm developed to expanded blastocysts as frequently (47%) as did good/intermediate oocytes (40%), but zygotes from oocytes with minimal cumulus developed less frequently (23%; P

Progress on gene transfer in farm animals

Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.

Flow cytometric sperm sorting and PCR to confirm separation of X‐ and Y‐chromosome bearing bovine sperm

Abstract The successful separation of X‐ and Y‐chromosome bearing sperm is dependent on the accurate assessment of their proportion in a population. In this study, their proportion was determined by molecular genetic analysis of hundreds of single sperm or by flow cytometric measurement of thousands of sperm. Separation was achieved by staining the DNA of bovine sperm with a viable dye, Hoechst 33342, to enable flow cytometric resolution and sorting of purified X or Y sperm populations. The resulting proportions of X and Y sperm (sort purities) have routinely been determined by flow cytometric reanalysis of the sorted sperm for their DNA content and subsequent curve fitting. Additional sort purity validation can now be achieved through molecular genetic analysis of single sperm. In this report individual X‐ and Y‐bearing intact sperm, prepared from neat semen, were sorted based on their total DNA content. The highly conserved zinc finger allele which resides on both the X and Y chromosome (zfx and zfy) wa...

Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype

The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells.