shRNA Knockdown of Chick MID1 and MID2 Using Recombinant Lentivirus

Cleft Lip and Palate (CLP) is a common midfacial disorder in which the developing facial processes fail to fuse. CLP can occur sporadically in a non-syndromal form, or it can be inherited in a Mendelian fashion. CLP is a common phenotype in the X-linked form of Opitz syndrome, which has been attributed to mutations in the MID1 gene. We are interested in understanding the role of MID1, and its partially redundant homolog MID2, in midfacial morphogenesis and the development of CLP. Objective: To study the involvement of MID proteins in the formation of CLP, we will use the developing chick embryo as a model. We aim to study MID function via knockdown of gene expression through the application of recombinant lentiviruses that encode shRNA taregeted against MID1 or MID2. Methods: We cloned previously tested shRNAs into a lentivirus vector and produced recombinant lentivirus using Invitrogen's ViraPower Lentiviral expression system. We assayed lentiviral knockdown of chick MID1 or MID2 transcripts using Invitrogen's BLOCK-iT assay. Cos-1 cells were transduced with viral stocks. Post-transduction, cells were then transfected with target sequence, MID1 or MID2, which was fused to LacZ. The efficacy of lentiviral, shRNA knockdown of MID1 or MID2 mRNA was measured by ß-Galactosidase readout. Results: Positive control plasmids expressing chick MID1 and MID2 shRNAs effectively reduced ß-Galactosidase activity by more than 90%, while unexpectedly, cells transduced by lentivirus showed an increase in ß-Gal activity over that of the control. Conclusions: While it appears that the recombinant lentiviral particles failed to knockdown expression of the transfected target gene, low viral titers, high transfection efficiency to low transduction ratios, and/or experimental design may have contributed to the unexpected result. To validate the use of recombinant lentivirus to knockdown gene expression, further testing will be necessary. Supported by T32DE007132.