Chat
Recent Searches
Part of Thermo Fisher Scientific  
 
 
 
Home |  RNAi | MicroRNA-adapted Retroviral Ve | Single copy knockdown with LMP

Single copy knockdown with LMP
Transcription of shRNAmir from RNA Polymerase II (Pol II) promoters is sufficient for highly effective knockdown of a target gene, even when expressed at single copy. This feature is essential for RNAi screens using complex libraries where infected cells are unlikely to contain multiple copies of a given shRNAmir vector.

To determine whether the LTR promoter, a strong Pol II promoter was sufficient for effective gene knockdown using shRNAmirs, an shRNAmir to p53 (p53.1224) was introduced into the LMP (MSCV/LTRmiR30-PIG) vector. To facilitate comparison,  LUMP (U6 promoter/miR-30PIG)-p53, UMP (U6 promoter, miR-30PIG-SIN-LTR)-p53 were introduced into NIH3T3 cells at a low multiplicity of infection (<5% efficiency) such that most transduced cells contained single or low copy proviral integrations. Both vectors carrying the MSCV-LTR supressed Trp53 extremely efficiently and were superior to UMP-p53.1224 which expresses p53 from the U6 promoter alone (from Dickens et al 2005).
 
Figure 1) Western blot analysis for Trp53 expression in NIH3T3 cells transduced at less than 5% efficiency (assessed by GFP FACS)  with LMP and other retroviral Pol II vectors

 

 
 
 
References:
Dickens NG et al (2005) "Probing tumor phenotypes using stable and regulated synthetic microRNA precursors" Nature Genetics Vol 37, No 11 1289-1295.