Self-inactivating lentiviruses: versatile vectors for quantitative transduction of cerebellar granule neurons and their progenitors.
Cerebellar granule neurons (CGNs) undergo a well-defined, intrinsic differentiation program that is recapitulated in vitro. Thus, homogeneous cultures of CGNs provide an excellent opportunity to define the mechanisms underlying their development. The ability to alter endogenous gene expression in CGNs on a population-wide basis would greatly facilitate the elucidation of these events. In the present study, we show that self-inactivating lentiviruses efficiently infect both dividing progenitors and post-mitotic CGN cultures in a quantitative manner without altering their cellular properties. The time course for protein expression was biphasic for both types of cultures, with the first peak occurring during the initial infection period. Thus, lentiviruses can express proteins in CGNs both acutely and on a long-term basis to study developmental and other processes continuously over an extended time period. These vectors also infected CGNs in cerebellar slice preparations. In addition, lentiviruses harboring a transgene for the mouse GABA(A) receptor alpha6 subunit promoter recapitulated the differentiation-dependent expression of this gene in CGN cultures. Self-inactivating lentiviruses are extremely versatile vectors that offer important advantages for studies of protein function and gene regulation. The ability to alter protein function on a global scale in CGN cultures permits biochemical assessment of its impact on mRNA and protein populations, as well as on protein--protein and protein--DNA interactions. Further, integrated lentiviruses can be used to study chromatin-dependent promoter regulation and transcription factor interactions in CGNs over time in a facile manner.
Wang
Wei
W
Department of Molecular and Cellular Physiology, University of Massachusetts Medical School, Basic Science Building, BSB S4-139, 55 Lake Avenue North, Worcester, MA 01655, USA.
Qu
Qiang
Q
Smith
Frances I
FI
Kilpatrick
Daniel L
DL
D000818:Animals; D002531:Cerebellum / Q000166:cytology / Q000821:virology; D015870:Gene Expression; D005822:Genetic Vectors; D016086:Lentivirus; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000821:virology*; D009924:Organ Culture Techniques; D011401:Promoter Regions, Genetic; D013234:Stem Cells / Q000166:cytology / Q000821:virology*; D014161:Transduction, Genetic / Q000379:methods*