L'Huillier P J, Soulier S, Stinnakre M G, Lepourry L, Davis S R, Mercier J C, Vilotte J L
Laboratoire de Génétique Biochimique et de Cytogénétique, Institut National de la Recherche Agronomique, Centre de Recherches de Jouy-en-Josas, France.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6698-703. doi: 10.1073/pnas.93.13.6698.
Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.
构建了在小鼠乳腺肿瘤病毒长末端重复序列转录控制下携带牛α-乳白蛋白（α-lac）特异性核酶基因的转基因小鼠，并将其与高表达牛α-lac转基因（每毫升乳汁中含0.4毫克α-lac）的动物进行杂交。该核酶含有锤头状催化结构域，两侧是与牛α-lac转录本3'非翻译区互补的12个核苷酸序列。通过Northern印迹分析在7 - 8日龄泌乳转基因小鼠的乳腺中检测到12个分析品系中有3个品系的核酶基因高水平表达。核酶的杂合表达导致三个独立品系的靶mRNA水平分别降至非核酶转基因同窝对照的78%、58%和50%。核酶介导的牛蛋白水平降低与mRNA的情况相似，并且与核酶的表达水平呈正相关。在不表达核酶的转基因小鼠中，牛α-lac mRNA和蛋白的水平不受影响。内源性小鼠α-lac mRNA或蛋白水平未降低，证明了这种活性的特异性。这些结果证明了核酶介导的转基因动物中高表达转录本下调的可行性。
