4.2. Generation and Transduction of Lentiviral Vectors

pGIPZ lentiviral vectors constitutively expressing an shRNA against the murine insulin receptor (mIR) (shRNA-mIR, CLONE ID: V2LMM_176881, V2LMM_176884, V2LMM_75164, V2LMM_79169, V3LMM_438244, V3LMM_452879) or the non-silencing (NS) control (RHS4346) were purchased from Dharmacon. Doxyciclyne-inducible pTRIPZ lentiviral vectors expressing shRNA-mIR (pTRIPZ-shRNA-mIRs) and shRNA-NS (pTRIPZ-shRNA-NS) were obtained following the manufacturer’s protocol (Dharmacon).

Doxyciclyne-inducible pTRIPZ-hIRA-FLAG and pTRIPZ-hIRB-FLAG lentiviral vectors were cloned from RG215257-hIR-A or RG215691-hIR-B plasmids (all from Origene) employing the following primers containing the FLAG sequence in the reverse primer:

Fw -hIRA and -hIRB:

5′-CGCACCGGTGCCACCATGGCCACCGGGGGCCG-3′;

Rv -hIRA and -hIRB:

5′-CGACGCGTCCTAGGTAATACGACTCACTATAGGGTTACTTATCGT

CGTCATCCTTGTAATCGGAAGGATTGGACCGAGGC-3′.

The obtained cDNAs were ligated in pTRIPZ vectors using the Age-I and Mlu-I restriction sites. Recombinant lentiviruses were produced, concentrated and used for transduction as previously described [20].