Fig. 1.

An shRNA-Encoding Lentivirus Allows for Efficient Silencing of the MR

A, Schematic representation of pDR-siMR used to express the MR-specific shRNA and the DsRed protein. B, Analysis of MR-expressing HeLa cells transduced with pDR (first lane) or pDR-siMR (second lane); normal HeLa cells served as a control (third lane). MR mRNA levels were studied by PCR (left panel) and MR protein levels by Western blot (right panel). β-Actin mRNA (Act) and β-tubulin (Tub) protein expression were used for normalization. C, HeLa cells were transduced with pDR or pDR-siMR, and two individual samples each were analyzed by Northern blot for the expression of the processed shRNA. A sense and an antisense oligonucleotide corresponding to the 19-nucleotide MR-specific siRNA as well as an oligonucleotide corresponding to the 5S rRNA were used for hybridization. D, PCR analysis of genomic DNA derived from the nine founder rats for the presence of the pDR-siMR provirus and the Notch1 gene as a control. CMV, Cytomegalovirus; LTR, long terminal repeat.