人红细胞生成素转基因小鼠乳腺生物反应器的建立

Production of Transgenic Mice Expressing hEPO in Mammary Gland

将 ２．４ ｋｂ 的人红细胞生成素（ｈ Ｅ Ｐ Ｏ）基因组基因（ｇ Ｄ Ｎ Ａ）ｍ ｉｎｉｇｅｎｅ 克隆于 ０．９７７ ｋｂ 大鼠乳清酸蛋白（ Ｗ Ａ Ｐ）５′调控序列下游和 ０．８５ ｋｂ Ｗ Ａ Ｐ３′侧翼序列上游，构建了 ｈ Ｅ Ｐ Ｏ 乳腺定位表达载体 ｐ Ｗ Ａ Ｐ Ｅ Ｐ Ｏ Ｗ Ａ Ｐ３′ Ｕ Ｔ Ｒ（ｐ Ｗ Ｅ３′）。载体经 Ｓａｃ Ｉ Ｉ酶切、回收并纯化后作为目的基因，通过显微注射方法导入小鼠受精卵的雄原核，共注射 ５００ 枚卵，移植 ３００ 枚卵至 ２９ 只假孕母鼠输卵管内，获得仔鼠 ５５ 只。采取鼠尾，提取基因组。经 Ｐ Ｃ Ｒ 检测，阳性鼠 ２２ 只； Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ 检测，阳性鼠 １６ 只，其中 ９ 只母鼠，７ 只公鼠。将 １６ 只首建者小鼠分别与非转基因鼠交配，共获得 Ｆ１ 代仔鼠 １５７ 只。应用 Ｐ Ｃ Ｒ 方法对 Ｆ１ 代小鼠进行检测，结果首建者１ 号母鼠所生的 ６ 只仔鼠中有 ３ 只为阳性。与此同时，于 ９ 只雌性首建者分娩后第 １０ 天采奶，应用 Ｅ Ｌ Ｉ Ｓ Ａ 方法对乳汁中的 Ｅ Ｐ Ｏ 进行检测，结果 ６ 只母鼠获得表达，表达量为 ０．１２～１．５９ μｇ／ Ｌ。

hEPO transgenic mice were generated by microinjection of a Sac Ⅱ linear sequence restriction fragment of the pWE3′Vector containing 0.977 kb of WAP 5′, 0.85 kb of WAP 3′ flanking sequence and 2.4 kb of hEPO genomic DNA minigene into fertilized eggs. Three hundreds of five hundred fertilized eggs microinjected were transplanted into twenty nine pseudopregnant mice. Eleven of the twenty nine pseudopregnant recipient mice became gestation and fifty five offspring were produced. PCR and Southern blotting techniques analyzed the genomic DNA samples extracted from the fifty five offspring tail. Sixteen of them including seven males and nine females were transgenic mice. Founders were mated with the nontrangenic mice respectively. One hundred and fifty seven offsprings were produced from the sixteen founders. Genomic DNA samples from the offspring were screened by PCR and three of six offspring from one female founder were transgenic mice. The others were nontransgenic mice. The milk collected on the tenth day after the nine female founder′s parturition was screened by ELISA. The results showed that six of nine female founders have expressed the hEPO protein in their milk and the expression level was at range of 0.12 1.59 μg/L.