Matsuda Takahiko, Cepko Constance L
Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):1027-32. doi: 10.1073/pnas.0610155104. Epub 2007 Jan 5.
In vivo electroporation is a powerful technique for the introduction of genes into organisms. Temporal and spatial regulation of expression of introduced genes, or of RNAi, would further enhance the utility of this method. Here we demonstrate conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain. For temporal regulation, Cre/loxP-mediated inducible expression vectors were used in combination with a vector expressing a conditionally active form of Cre recombinase, which is activated by 4-hydroxytamoxifen. Onset of gene expression was regulated by the timing of 4-hydroxytamoxifen administration. For spatial regulation, transgenes were expressed by using promoters specific for rod photoreceptors, bipolar cells, amacrine cells, Müller glia or progenitor cells. Combinations of these constructs will facilitate a variety of experiments, including cell-type-specific gene misexpression, conditional RNAi, and fate mapping of progenitor and precursor cells.
体内电穿孔是一种将基因导入生物体的强大技术。对导入基因或RNA干扰的表达进行时空调控,将进一步提高该方法的实用性。在此,我们展示了在出生后大鼠视网膜和胚胎小鼠脑中对电穿孔质粒基因表达的条件性调控。对于时间调控,Cre/loxP介导的诱导型表达载体与表达由4-羟基他莫昔芬激活的条件性活性形式Cre重组酶的载体联合使用。基因表达的起始由4-羟基他莫昔芬给药时间调控。对于空间调控,通过使用视杆光感受器、双极细胞、无长突细胞、穆勒胶质细胞或祖细胞特异性的启动子来表达转基因。这些构建体的组合将有助于开展各种实验,包括细胞类型特异性基因错误表达、条件性RNA干扰以及祖细胞和前体细胞的命运图谱绘制。