Establishment Of Transgenic Goats For Human Tissue-type Plasminogen Activator With Sperm Mediated Gene Transfer Method

The production of valuable human pharmaceuticals in large farm animals (pig, sheep, goat and dairy cattle) has become more and more attractive as a high-quantity, low-cost alternative. We know that production of mammary gland bioreactors depend on the availability of reliable techniques to obtain transgenic animals with the necessary spectrum of transgene(s) expression.Today, transgenic animals have been produced for the most part by using microinjection of exogenous DNA into the male pronuclei of zygote. This technique, although highly successful in mice, is not as efficient in farm animals, limiting its general utility.Compare to others transgenic techniques, sperm mediated-gene transfer (SMGT) has two additional advantages: low cost and easy of use. However, the availability and reliability of SMGT hasn't been approbated. In our study, we investigated the prosess of exogenous DNA binding and internalization to sperm and the molecular events of exogenous DNA after internalization. Our aims were to establish the platform of SMGT and use the technology system to produce transgenic goats for human tissue-type plasminogen activator. Results as following:1) Goat spermatozoa could spontaneously take up exogenous DNA. There were considerable differences in the capability of spermatozoa from different donors to bind and internalize exogenous DNA. For the spermatozoa from the same goat, the binding and internalization capacities were mostly inhibited by the seminal fluid. Compared to ejaculate sperm, the binding rate and internalization rate were increased several times in washed sperm cells.2) Dead spermatozoa did not complete the internalization process. The highest positive rate (before DNase I digestion) was found in membrane-broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors.3) Factor(s) in seminal fliud reacted with DNA and formed DNA complex or digesteddissociated DNA but inhibited by EDTA. DNA complex concentration and digestion effect were dependent on seminal fliud concentration and incubation time.4) Most internalized DNA was digested and its effect was inhibited when spermatozoa incubated with EGTA/ATA for 30min.5) After co-cultured with pEGFP-N1 plasmid DNA, spermatozoa were used to fertilized with oocytes. Report gene GFP expressed in embryos. Frequencies of GFP expression and positive embryos tested by PCR were correlated with the capability of spermatozoa to take up exogenous DNA6) Goat spermatozoa, incubated with 10 μg/mL pBGC-mtPA plasmid DNA for 45~60min, used by artificial insemination to yield transgenic animals. 129 kidlets were identified by PCR and Southern blotting. 24 out of 129 (18.6%) were positive for pBGC-mtPA tested by PCR and 15 kidlets (11.6%) were positive tested by Southern blotting. However, pregnancy rate reduced and oafish kidlets increased when transformed spermatozoa were used for insemination.