RGC–Targeted Gene Knockdown in Adult Conditional Knockout Mice Using AAV–Mediated Intravitreal Delivery of Cre Recombinase

Abstract

Abstract: : Purpose: The use of conditional gene targeting has led to approaches controlling gene knockouts in vivo. Previous studies have shown that adeno–associated virus (AAV) vectors efficiently deliver functional Cre recombinase to the mouse CNS in vivo, without requiring the generation of transgenic mice expressing inducible Cre. We determined if AAV could deliver Cre to the retina, providing a means to perform gene knockouts specifically in retinal ganglion cells (RGCs) of adult conditional knockout mice. Methods: We obtained AAV constructs from Dr. William Hauswirth (University of Florida, Gainesville, USA). We and others have shown that AAV constructs using the chicken ß–actin (CBA) promoter express genes of interest extremely well in rodent RGCs. Further enhancement of AAV–mediated gene expression is achieved by adding the woodchuck post–transcriptional regulation element (WPRE) to the 3’ end of the viral construct. We employed this vector to express Cre in retinas of Rosa26 reporter mice. We performed intravitreal injections of 3 µl of AAV–Cre in one eye of Rosa26 mice. After four weeks to allow expression of Cre, we performed X–gal staining on sections from the AAV–injected and paired control eyes. H&E staining was then done to identify ganglion cell nuclei. Results: Retinas transfected with AAV–Cre showed extensive ß–galactosidase activity, demonstrating that Cre–mediated knockout of the negative regulatory element in the Rosa26 locus had allowed ß–galactosidase gene expression. X–gal positive cells were noted primarily in the RGC layer, and to a lesser extent in the inner nuclear layer. Control retinas (no AAV–Cre injection) showed no ß–galactosidase activity. Comparison of the number of X–gal positive cells and H&E stained cells in the ganglion cell layer showed that approximately 70% of cells were transduced with AAV–Cre, consistent with previous reports using this vector construct. Conclusions: This represents the first report of retina–specific gene knockdown in adult conditional knockout mice using AAV delivery of Cre. It is now possible to perform RGC–specific gene deletions in adult conditional knockout mice that have been unsuitable for experimentation due to embryonic lethality or premature death in the standard knockout mice.

Keywords: ganglion cells • gene transfer/gene therapy • apoptosis/cell death