Tashiro Ayumu, Zhao Chunmei, Gage Fred H
Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Nat Protoc. 2006;1(6):3049-55. doi: 10.1038/nprot.2006.473.
Single-cell genetic manipulation in an intact brain environment is an informative approach to study molecular mechanism of adult neurogenesis. Here, we describe a protocol for retrovirus-mediated single-cell gene knockout in adult new neurons in vivo. A gene of interest is disrupted in adult floxed mice by a vector based on the Moloney murine leukemia retrovirus, expressing Cre recombinase. High-titer retrovirus is prepared by transfecting plasmids into the HEK293T cells and by concentrating the supernatant containing virus. The retrovirus is stereotaxically injected into the dentate gyrus. Cre recombinase is transduced and expressed in a small fraction of adult new neurons in an intact environment, and the gene knockout is highly efficient within the transduced neurons. Virus preparation takes 7 days, but virus injections take less than 1 h per mouse. By changing the survival time of the mice after the injection, one can analyze the effects on new neurons at different ages.
在完整的脑环境中进行单细胞基因操作是研究成体神经发生分子机制的一种信息丰富的方法。在此，我们描述了一种在成年小鼠体内新生成的神经元中通过逆转录病毒介导进行单细胞基因敲除的方案。通过基于莫洛尼鼠白血病逆转录病毒的载体表达Cre重组酶，在成年的floxed小鼠中破坏感兴趣的基因。通过将质粒转染到HEK293T细胞中并浓缩含有病毒的上清液来制备高滴度逆转录病毒。将逆转录病毒立体定向注射到齿状回。Cre重组酶在完整环境中的一小部分成年新生成的神经元中被转导并表达，并且在转导的神经元内基因敲除效率很高。病毒制备需要7天，但每只小鼠的病毒注射时间不到1小时。通过改变注射后小鼠的存活时间，可以分析对不同年龄新生成神经元的影响。
