Gene Transfer in The Porcine and mouse Embryos

本研究計畫旨在於應用顯微注射技術，將含有牛α-乳白蛋白基因(α-lactoalbumin ; αLA)調節序列與人類凝血第九因子(human blood coagulation factor IX;hFIX)基因構造性cDNA序列，長度為4.93 kb之αLA-hFIX 轉殖基因片段，注入小鼠與豬之早期胚原核中，並將完成基因注射後之小鼠胚與豬胚，分別移置於受胚小鼠與受胚豬之生殖道內，冀能從而產生帶有αLA-hFIX之基因轉殖小鼠與基因轉殖豬。試驗結果證 實已成功獲得14 隻(2♀/9♂+3?)基因轉殖小鼠及2 頭(1♀/1♂)基因轉殖豬。進一步將此等基因轉殖小鼠及豬分別與非基因轉殖者進行配種後，不僅證明其中9 隻(9♂)小鼠及2頭(1♀/1♂)基因轉殖豬係具有生殖能力者，且均可將轉殖之αLA-hFIX 基因分別遺傳至其子代。于哺乳期間分別收集基因轉殖小鼠及豬之乳汁樣品，並進行Western blot 及凝血活性分析，結果已證明五個家族之基因轉殖小鼠及一個家族之基因轉殖豬之乳汁中均含有αLA-hFIX 基因之轉譯物，依酵素免疫分析法定量之結果顯示濃度分別約為50-200μg/ml 及200-500 μg/ml，並證實均具促使缺hFIX 血漿凝血之活性。此外，將性成熟之第0 代基因轉殖公豬，採新鮮精液或製備冷凍精液，並以人工授精方式與非基因轉殖藍瑞斯(L)種母豬配種亦可獲得攜轉殖基因之後代。顯示，基因轉殖公豬精液可採用冷凍保存精液方式進行保種。此結果提示，基因轉殖小鼠及豬之乳腺可表現具生物活性之醫藥蛋白，未來將考慮及評估以豬乳腺量產hFIX之可行性及經濟性。The purpose of this study was to generate transgenic mice and piglets by method of microinjection the αLA- hFIX gene, a 4.93 kb fragment contained the regulatory sequences of bovineα-lactoalbumin and the structure sequence of cDNA from human factor IX, into the pronucleus of newly fertilized mouse and porcine eggs, respectively. Experimental results showed eleven (2♀ / 9♂) newborn mice and two (1♀ /1♂) of the newborn piglets were confirmed to be as transgenics, according the results from PCR and Southern-blot analysis which had been subjected to PCR and Southern-blot analysis were comfirmed to be successfully integrated with the transgene. The transgenic founders mated with nontransgenic mice and pigs, respectively, found the transgene stably integrated in the germ line. Detecting by Western-blot analysis confirmed that expression of αLA-hFIX transgene with hFIX concentration between 50-200μg/ml and 200- 500μg/ml contained in the milk from lactating transgenic mice and sow, respectively. The hFIX APTT clotting activity was assayed directly on milk collected from transgenic mice and pig, the data showed that the hFIX synthesized by transgenic mice and pig is active. Furthermore, frozen semen of L-8-5 were prepared and inseminated to nontransgenic sow, could transmit the transgene to progenies, demonstrating that the transgenic line can be preserved by frozen semen. In the future, focusing our attention on economic consequences and product safety may be important