994. Gene Knockdown in Mice by Using Lentiviral Vectors Expressing Small Interfering RNA

Top of pageAbstract Many approaches have been described recently for generating loss-of-function phenotypes in eukaryotic systems using RNAi. Use of RNAi by means of long dsRNAs (>50bp) is limited in mammalian cells due to the induction of a nonspecific inhibitory response resulting from the interferon pathway. This limitation was overcome by the finding that small interfering RNAs (siRNAs) can specifically silence genes in mammalian cells without activating the nonspecific interferon pathway. We have designed lentiviral vectors expressing siRNAs to knock down the expression of specific genes. We have previously shown that a lentiviral vector capable of expressing siRNA specific for GFP (siGFP) was able to silence GFP expression in TgGFP mice upon transduction of fertilized mouse embryos. We have found that transgenesis of mouse embryos can be achieved both by zona pellucida removal prior to transduction and by microinjection of lentivirus particles under the intact zona layer. Efficiency of transgenesis was high using both methods, up to 80% and 100% respectively. However, survival of the treated embryos was low in the case of zona removal techniques (10-20%), compared to 40-60% for microinjection. We are currently using transgenesis by lentiviral vectors expressing small interfering RNAs to generate knockdown mice to study functional genomics.