Transgenic animals are generated by injection of recombinant DNA sequences into fertilized oocytes. Here I applied a new methodology for the generation of transgenic knockdown mice using the LentiLox 3.7 lentivirus as a transfer vehicle bearing an U6-promoter dependent shRNA expression cassette. Lentiviruses are a sub-class of retroviruses that have the capability to infect non-dividing post-mitotic cells. Recently, in addition to their use to transform primary cells and established cell lines, lentiviruses have also been used to generate transgenic mice, pigs, cattle, rats and chickens. Thus, we hoped that lentiviral vectors containing U6/RNAi expression cassettes could serve as a fast and attractive alternative for the generation of mice with reduced expression of specific genes. As a model for this in vivo knockdown approach I chose the hepatocyte nuclear receptor 4gamma for which a knock out model was not available. Expression analyses of the HNF4gamma gene demonstrated synthesis of the protein in the embryonic gut at day E16.5. In adult animals its expression is restricted predominantly to the differentiated, absorptive brush border cells of the small intestine (enterocytes) and to the cells of pancreatic islets (islets of Langerhans). In order to knockdown the HNF4gamma gene, a panel of five shRNA hairpin sequences was selected by the public Sirna software and their activity was validated by transfection experiments in cell culture. After re-cloning of the U6/shRNA cassettes into the pLL 3.7 vector, infectious virus particles were generated and injected within the perivitelline space of one cell stage mouse embryos. 56% of the LentiLox 3.7 lentivirus founder mice were PCR-positive, however expression from the transgene was highly mosaic. The high mosaicism of F0 mice precluded their use for immediate expression analysis as it was hoped when the project was started. The high degree of mosaicism is also reflected by a low rate of germ line transmission. Only 6% of F1 mice expressed the indicator gene for EGFP as well as the shRNA transgene. Often expression was not ubiquitous probably reflecting the dependence of expression on the chromosomal integration site. A good correlation between EGFP activity and siRNA accumulation in organs of F1 mice was found as evidenced by Northern blot hybridisation. Despite the general low efficiency of transgenesis the down regulation of HNF4gamma gene in one F1 line (A-I) reached 50% in the gut and 80% in pancreas proving that this targeted knockdown approach is working in living animals.