An MSCV-Based shRNA Expression Vector for Mouse Hematopoietic Cells

At Cellecta, we have relied primarily on lentiviral vectors to develop our RNAi genetic screening technology. The broad tropism of lentivirus-based plasmids packaged into VSV-g pseudotyped viral particles provides a very convenient vector system to introduce and stably express shRNA expression constructs or constructs expressing small proteins, peptides, or other biological molecules into virtually any mammalian cell line. Unlike with most retroviruses, cells do not need to be dividing take up a lentivirus, and unlike adenoviruses, the lentiviral expression construct integrates into the cell's genomic DNA so it is stably passed onto progeny allowing long term studies over several cell passages. Also, in contrast with advenoviral vectors, lentiviral plasmids can be easily engineered allowing construction of complex pooled libraries such as those we use for RNAi screening.

However, there are some cells for which lentiviral vectors seem to work poorly. In particular, mouse hematopoietic cells appear to sometimes shut down expression of lentiviral constructs. We have found this anecdotally based on feedback from researchers using our system, and it has been noted in published literature. To address this, we looked into developing a more effective vector for stable shRNA expression in these types of cells.

We adapted a murine stem cell viral (MSCV) plasmid with our shRNA cloning cassette, which incorporates the U6 promoter to drive hairpin expression. As with our standard lentiviral constructs, the MSCV shRNA vector constitutively expresses a fluorescent protein marker (tagRFP) and puromycin antibiotic selection gene on a single transcript separated by a 2A self-cleaving peptide. These features enable easy assessment of infection rates and selection for transduced shRNA-expressing cells.

University of Pennsylvania uses Cellecta pMSCV-U6-sh-UbiC-TagRFP-2A-Puro vector system

To test knockdown, we cloned an shRNA known to effectively knock down mouse p53 into the MSCV plasmid. Vikram Paralkar in Mitch Weiss's lab at the Children's Hospital of Philadelphia tested the effectiveness of the construct in erythroid progenitor cells extracted from day 14.5 CD-1 mouse embryo fetal livers. The erythroid progenitors were infected with the MSCV-p53 construct, as well a control construct that did not contain the p53 sequence, packaged in viral particles. A multiplicity of infection (MOI) of virus to cells was used such that approximately 90% of the cells were transduced. Then, after 48 hours, the cells were harvested and RT-PCR was used to assess the expression level of the p53 target gene. The results below show that at least 75% knockdown of the p53 target transcript was obtained.

University of Pennsylvania shows shRNA knockdown of p53 in mouse hematopoietic cells using MSCV vector system

The results indicate that this MSCV vector provides an effective alternative for shRNA expression in mouse hematopoietic cells where lentivirus infection is problematic. The efficiency levels of cloning and transduction with the MSCV vector appear to be sufficient to enable construction and screening with complex pooled shRNA libraries as effectively as with our standard lentiviral system.

Leave a comment

Comments will be approved before showing up.

Also in Cellecta Blog & News

Inducible Cas9 Expression in a Single Lentiviral Vector

Introducing Inducible Cas9 Expression in a Single Lentiviral Vector to make cells capable of high Cas9 expression for a limited time during which CRISPR-mediated targeted rearrangements can occur, and then shut off Cas9 expression for downstream assays with the modified cells.
Read More
Insertion of 10X Genomics' Capture Sequences Does Not Affect HEAT-Tracr sgRNA Efficacy

Perturb-Seq or CROP-Seq screens make use of single-cell RNA-Sequencing in conjunction with a pooled CRISPR library to identify transcriptional changes and, by implication, activation or deactivation of cellular pathways related to phenotypic changes produced by specific sgRNA-mediated gene knockouts.
Read More
Core Population of Cancer Stem Cells Mediates Therapeutic Resistance in Tumors

Researchers at MD Anderson Cancer Center recently used a Cellecta CloneTracker Barcode Library to label patient-derived xenograft (PDX) cells and establish a stable population of aggressive tumorigenic cells with a specific set of barcodes. With this population of barcoded tumorigenic clones, the investigators...
Read More