Division of Animal Sciences, University of Missouri, 159 ASRC, 920 East Campus Dr., Columbia, MO, 65211, USA.
Transgenic Res. 2018 Apr;27(2):167-178. doi: 10.1007/s11248-018-0064-3. Epub 2018 Mar 7.
Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre-loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/μL when compared to 2 and 100 ng/μL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/μL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.
基因工程猪通常是使用含有loxP 侧翼选择标记（如新霉素）的靶向载体创建的。Cre-loxP 重组酶系统可用于从产生的后代或细胞系中去除选择标记基因。本文描述了一种通过直接向受精卵注射编码 Cre 重组酶的 mRNA 来去除loxP 侧翼新霉素盒的新方法。与 2 和 100 ng/μL（P<0.0001）相比，当比较 10 ng/μL 时，mRNA 的最佳浓度确定为 10 ng/μL。用 10 ng/μL 的 mRNA 注射受精卵后，发育到囊胚阶段的比例为 14.1%。该方法成功地去除了 81.9%的注射体外衍生胚胎中的新霉素盒；与对照组相比，这一比例显著更高（P<0.0001）。胚胎移植导致一只携带 Cre 删除的新霉素盒的活小猪出生。所描述的新方法可用于有效去除基因工程动物中的选择标记，而无需长期细胞培养和随后的体细胞核转移。
