Engineering viral promoters for gene transfer to human neuroblasts.
1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene constructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control. 3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts. 4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.
Cara
A
A
Department of Infectious Diseases, Mount Sinai School of Medicine, New York, New York, USA. acara@usa.net
Lucarelli
E
E
Cornaglia-Ferraris
P
P
D001358:Avian Sarcoma Viruses / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D009447:Neuroblastoma; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics; D020079:Terminal Repeat Sequences
D008156:Luciferases