Manipulation of Sperm for Efficient Production of Transgenic Calves and Chicks

The current method of micromanipulation used for domestic animals results in less than 1% transgenic animals. This makes it extremely difficult to produce transgenic cows and is not feasible for producing transgenic chickens. The purpose of this work was to find a more efficient method for producing transgenic calves and chicks using a combination of two techniques, lipofection and restriction enzyme mediated insertion (REMI). Previously investigators were unable to produce transgenic chickens using lipofection alone. On the other hand, injection of isolated sperm nucleus incubated with restriction enzyme into oocytes has only been shown to be effective in frogs. In this study, we demonstrated for the first time, that lipofection of both DNA and restriction enzyme could be used to successfully integrate DNA into the sperm genome DNA and then used for routine AI to produce transgenic calves and chicks. First it was demonstrated using needle pricking and southern blot analysis of genomic DNA that the restriction enzyme opens up “hot” spots in the sperm genomic DNA. This produces sticky ends by which foreign DNA can be inserted and integrated into the sperm genomic DNA. The “transgenic sperm” thus made were used in IVF and AI to produce embryos expressing a foreign DNA, EGFP (enhanced green fluorescent protein). Using Not I and linearized pEGFP lipofected to sperm for AI resulted with two calves which expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP protein; (c) homology analysis between EGFP DNA and PCR product DNA sequences and (d) Southern blot analysis. Similarly in the chicken, linearized plasmid EGFP sequences with the corresponding restriction enzyme (REMI) were lipofected into the sperm. The transfected sperm were then used for AI in hens and 90% (17/19) of the resultant chicks expressed the exogenous DNA in their lymphocytes as determined by: (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP; and (c) Southern blot analysis. A complete homology was found between the Jellyfish EGFP DNA and a 313 bp PCR product of DNA from chick blood cells. The procedure was then tested with an additional construct, hFSH. The construct of hFSH consisted of both subunits, α and β and the PCR product used primers for both α and β subunit resulted with a PCR product of 584 bp which was unique to transgenic chickens. The procedure was then used to lipofect a construct of hFSH (Human Follicular Stimulating Hormone) into chicken sperm and used for AI. The resultant offspring were transgenic for at least three generations as determined by: (1) measurement of hFSH protein in chicken blood using enzyme immunoassay and RIA; (2) RT-PCR and PCR; and (3) copy number.

We conclude: (1) that lipofection of both DNA and restriction enzyme into sperm (bovine and chicken) induces the integration of the DNA into the sperm genomic DNA; (2) lipofected sperm can be used in AI to produce a high percentage of transgenic calves and chicks; (3) The integrated gene is expressed in the first, second and third generation; and (4) the method is not limited to specific genes. The technique of lipofection of DNA combined with REMI is therefore an efficient and stable method of producing transgenic domestic animals. Efficient production of transgenic domestic animals could have major impact on gene therapy, improving livestock breeds and the production of valuable pharmaceuticals, e.g. hFSH, which could be extracted from eggs and milk.

Keywords: Lipofection, Lipofected sperm, Restriction enzyme, Transgenic chicken, Transgenic cow, hFSH, GFP, pDNA.