Use of a retroviral vector with an internal opsin promoter to direct gene expression to retinal photoreceptor cells.
OBJECTIVE
Viral-mediated gene transfer to retina, as well as to other tissues, is evolving rapidly. We have evaluated the potential of a retroviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells.


METHODS
Two recombinant retroviral vectors were prepared; in each Vector, a 1.4 kb fragment of the mouse opsin promoter was placed downstream from the neoR gene in the Moloney murine leukemia virus-based vector G1Na. The opsin promoter fragment was linked either to the cDNA for mouse rod photoreceptor phosphodiesterase (PDE) beta-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expression after transduction of 3T3 and Y79 cells, or of dissociated retinal cell cultures or retinal explants from neonatal mice.


RESULTS
As expected, PDE beta-subunit and beta-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. Northern blot analysis indicated that expression was derived from the viral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in which beta-galactosidase is driven by the viral LTR, resulted in expression in many cell types, while the opsin-lacZ vector mediated the expression of the lacZ reporter gene specifically in photoreceptor cells.


CONCLUSIONS
The internal opsin promoter fragment appears capable of selectively directing gene expression to photoreceptor cells after retroviral-mediated gene transfer. These findings serve as a basis for future studies using the opsin promoter-beta PDE retroviral vector to rescue photoreceptor cells in the rd mutant mouse, in which the beta-PDE gene is mutated resulting in degeneration of photoreceptor cells during the early postnatal period.
Kido
M
M
Doheny Eye Institute Department of Ophthalmology, USC School of Medicine, Los Angeles, CA, USA.
Rich
K A
KA
Yang
G
G
Barrón
E
E
Kohn
D B
DB
al-Ubaidi
M R
MR
Blanks
J C
JC
Lang
G
G
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D005134:Eye Neoplasms / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007124:Immunoenzyme Techniques; D007763:Lac Operon / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008979:Moloney murine leukemia virus / Q000235:genetics*; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D010786:Photoreceptor Cells / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D012160:Retina / Q000378:metabolism; D012175:Retinoblastoma / Q000378:metabolism; D017299:Rod Opsins / Q000235:genetics*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D017299:Rod Opsins; D010727:Phosphoric Diester Hydrolases; D001616:beta-Galactosidase