| Cunent gene transfer methods in transgenic animals include embiyo microzrijection, embiyonic stem cell mediated and retrovims mediated They have been videIy applied to construct transgeriic large animal as well as mice. However, when these methods were used to construct transgenic animals, there are some shortcomings, for example, time consuming and high cost In order to overcome these shortcomings we bad developed a new method to construct transgenic mice. Two important considerations had been taken into account, i.e. the gene transfer efficiency and the target cell. Recombinant retrovirus is selected as gene transfer method and spermtogonial stem cells were used as target cells. Recombinant retroviral vectors (pLLSN and pLNCL) cartying LazZ were constnLcted. The two vectors, together with pLNCX and pLXSN canying NeoR were transfected into PA3 17 for packaging by means of Lipofectin and calcium phosphate precipitation respectively. After G41 8 selection at a concentration of O.3g/L for 2 weeks, G41 8-resistant PA317 colonies were obtained and amplified, and four G41 8-resistant PA3 17 cell lines (PA-I, PA-2, PA-3, PA-4) were obtainecL The supernatant of the cell culture was harvested, concentrated and observed under transmission electron microscope, the mature vinis particles possessed the typically morphological feature of retrovirus. The concentrated viral supemantant fim PA-3 and PA-4 was used to infect Nll{3T3. The infected NIH3T3 cells were fixed by O.5(wlv) parafoima]dehyde and stained by X-gal, those cells that expressed LacZ became blue. The result showed LzcZ gene had been expressed on NIH3T3 cell. At the same time, Nll-13T3 cells were infected to measure the viral titer of recombinant retrovirus, Results showed that the titer of concentrated viral supematant from PA-2 was the highest, and was 3 X lO6cfu/ml. 84 In order to study the gene transfer mediated by recombinant retrovims- spermatogonial stem cells, recombinant reUuvirus from PA-2, canying NeoR, together with typan blue and Polybrene, were injected into contorted seminiferous tubule of 10 mice (14-16 day) to infect spermatogonial stem cells in vivo. After 40 days, sperms were harvested from injected mice. Its qualities( living mtio. apical body intact ratio and deformation ratio) were examined, the data were collected and statistically analyzed. PCR was used to examine the integration of NeoR the genome of sperm. The results showed that injection solution doesn抰 have conlraiy effect on the development of sperm. NeaR gene were confirmed to be integrated into the genome of mature sperms from eight mice. Female mice were mated with the eight male mice respectively, and forty-eight ofipring were produced. PCR and Southern blotting were used to screen NeaR gene from the genomic DNA samples extracted from the forty-eight olpring tails. Four of them including one male and three females were demonstrated to be lransgenic. Four founders were further mated with the nontransgenic mice and fifceen offspring were produced and PCR were used to screen NeOR gene integration. Four out of five offspring from one female founder were transgenic mice. Theinteonratewas8.3%(4/48),andwasalmostastheeashaven reported. But gene transfer mediated by retrovirus-spemitogonial stem cell is much easier to manipulate than other methods and it saves time. So, gene transfer mediated by recombinant retrovinis - sperrnatogonial stem cells appears to be a promising way to construct transgenic animals, such as mice as well as large animals.
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