Clp1 knockdown lentigenic mouse generation

Clp1 promotes 3'UTR shortening and higher levels of Aire-upregulated transcripts in mTEChi Overall design: Three shRNAs against Clp1 were cloned into a cluster of micro RNAs construct. This construct was transferred to a lentiviral backbone, downstream of the doxycycline inducible promoter TRE3G and upstream of the ZsGreen protein. A second construct expressing the TetOn3G transactivator under the control of the EF1 promoter was generated. A single ultra-high purified and concentrated lentivector (2.2x109 TU/mL) containing both constructs was generated and purified by both Tangential Flow Filtration and Chromatography. Fertilized oocytes (B6) were microinjected under the zona pellucida with the lentivirus suspension. Transduced oocytes were reimplanted into pseudopregnant females. Newborns were selected for integration of both constructs by PCR with primers matching the ZsGreen or TetOn3G sequences. At 3 weeks of age, mice were treated with doxycycline food pellets (2 g/kg) for two weeks and then sacrificed for mTEChi isolation. One generated pup, with integration of both plasmids. exhibited, after doxycycline treatment, a 60% reduction of Clp1 mRNA levels in GFP+ (Clp1 knockdown) versus GFP- (Ctr) mTEChi taken from the same mouse. Total RNA isolated by Trizol extraction to generate poly-A-selected transcriptome libraries using the Smarter Ultra Low Input RNA kit (Clontech) combined to the Nextera library preparation kit (Illumina). Single-end sequencing was performed on the Illumina NextSeq 500 machine.