Alternative methods to produce transgenic animals

Genetic engineering techniques have allowed the production of recombinant proteins in heterologous species. Transgenic mice, goats, sheep and pigs have been shown to be capable of producing heterologous proteins. One of the objectives in producing transgenic livestock is the large scale production of bioactive pharmaceutical and other valuable proteins. Secretion of proteins in milk is one of the methods for efficient mass production of recombinant proteins in cattle. The expression of bioactive recombinant proteins in the mammary gland of transgenic goats and sheep is well established. Dairy cattle would be the preferred animal for mass production of heterologous proteins, because of the high protein yield in milk. Many laboratories have attempted to produce transgenic cattle by pronuclear injection of recombinant DNA into fertilized oocytes. This method has been widely applied in mice but has comparatively low efficiency in cattle. Different methods have been tried to improve the efficiency of transgenic cattle production. The most commonly used method is the combination of biopsy technique and PCR analysis of preimplantation embryonic tissue. However, the high false positive rate of PCR analysis and the low transgenic rate in animals born indicate the inadequacy of this method. Here we target critical steps in various upstream and downstream methods to improve transgenic cattle production. These include two non-invasive transgenic reporters: the secreted placental alkaline phosphatase and the green fluorescent protein, and a novel gene transfer system: VSV-G pseudotyped retroviral vector. These non-invasive transgenic reporters do not require the biopsy of preimplanation embryonic tissue. A moderate transgenic embryo selection accuracy of 60-70% was demonstrated in using SEAP as a transgenic reporter. The expression of GFP in preimplantation embryos not only allows the accurate selection of transgenic embryos but also alleviate the problem of transgenic mosaicism. The VSV-G pseudotyped retroviral vector allows an efficient delivery of foreign DNA into metaphase II arrested oocytes and early stage embryos. Successful production of transgenic cattle with an efficiency approaching 100% indicates the potential application of this novel gene transfer system which allows the practical use of transgenic technologies in farm animals.