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.
