Expression of transgenes in primary neurons from chick peripheral and central nervous systems by retroviral infection of early embryos.
Many cell biological studies require the expression of transgenes in cells in culture, but it is difficult to obtain uniform, stable, and efficient expression of transgenes in primary neurons. We have approached this problem by adapting from developmental biologists the avian retroviral vector, RCAS. This vector allows the introduction of a transgene by infection early in chick embryonic development. Transgenes that are less than 2.6 kb in size can be cloned through an adapter vector, SLAX 12 NCO, and into the RCAS retroviral vector with relative ease. The vector is then used to produce active virus, and the virus is injected into the neural tube or ventricles of stage 10 embryos. By infecting the neuronal precursor cells while they are still mitotic, the retrovirus and accompanying transgene are introduced into the genome and subsequently spread by replication, shedding of new virus, and infection of other cells. Embryos are incubated from the time of injection until E9-E12 and peripheral and central nervous system neurons are dissected out and grown in culture using standard techniques. In this manner, the majority of the sympathetic and dorsal root ganglion neurons can be induced to express the trangene. A similar result, at lower efficiencies, is obtained for central nervous system neurons.
Hollenbeck
Peter J
PJ
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Fekete
D M
DM
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002642:Chick Embryo; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D009420:Nervous System / Q000166:cytology / Q000821:virology; D009474:Neurons / Q000166:cytology / Q000502:physiology / Q000821:virology*; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000295:instrumentation / Q000379:methods*; D019076:Transgenes / Q000235:genetics*