pLKO-1 Vector & shRNA Design

Is pLKO.1 vector a HIV-based vector and if so are there any bio-safety issues?

How do the features of the viral system work?

Why is the U6 promoter used?

What restriction sites were used for cloning the shRNA sequences? Do I need to transfer the sequences into a different vector backbone?

Can I search and design my own shRNAs on the TRC "Search for shRNA hairpin sequences page"?

Why doesn't the non-target control vector affect mammalian gene expression?

Can the MISSION^® lentiviral particles be further propagated in the lab?

Why is there always a clone for the 3' UTR region?

Which packaging systems are compatible with pLKO.1 shRNA vectors?

Does the MISSION^® TRC Library include a bar code that I could use for microarray analysis?

The protein I intend to knockdown is very stable, does the expression of the siRNA extend over a long period, where the cells are dividing?

Does pLKO.1 contain WPRE?

MISSION^® shRNA Vector Map

Is pLKO.1 vector a HIV-based vector and if so are there any bio-safety issues?

The pLKO.1 vector is a lentiviral (HIV)-based plasmid. The vector is regarded as a bio-safety level 2 material and safe to use due to its modified features (deletion of a number of accessory genes implicated in the virulence of HIV, minimal genome of the viral particles, non-replicating and self-inactivation features), making it incapable of producing virus once infected into the host cell. Please consult with your institution’s biosafety officer on specific requirements.

Can the MISSION^® lentiviral particles be further propagated in the lab?

No, the viral particles cannot be propagated for biosafety reasons. The MISSION^® TRC lentiviral particles are replication incompetent. The particles are made using features of 2^nd and 3^nd generation lentiviral packaging systems. Genes for replication and structural proteins are absent in the packaged viral genome since these genes are supplied by other plasmids in the packaging cells. The viral genome contains only the region between the 5’ and 3’ LTRs of pLKO.1. In addition, the lentiviral vector contains a self-inactivating 3’ LTR that renders it unable to produce infectious virus once it integrates into the host chromosome.

How do the features of the viral system work?

The library is lentiviral based. This allows for the production of viral particles in an appropriate packaging cell line. Upon infection with the resulting lentiviral particles, the shRNA sequence is integrated into the chromosome for stable expression of the hairpin RNA. The VSV-G envelope is pantropic and allows delivery to virtually any cell. In addition, lentivirus does not require a mitotic event for integration into the host cell genome.

Why is there always a clone for the 3' UTR region?

The clones that are against the 3' UTR of the target gene are included so one could rescue the knockdown phenotype with the introduction of a cDNA clone corresponding to the target. Transcripts from an exogenous cDNA clone would not be susceptible to silencing compared to the endogenous copy. Many publications/review committees require such an experiment to verify the observed phenotype. However, while most gene sets have this added feature, not all of them have a 3' UTR clone.

Why is the U6 promoter used?

The human U6 promoter (a pol III promoter) is used to drive expression of the shRNA hairpin. Expression using pol III promoters is optimal for producing shRNAs due to precise initiation and termination of transcription. TRC chose U6 based on experimental data showing excellent efficiency when compared to another efficient pol III promoter (H1).

Which packaging systems are compatible with pLKO.1 shRNA vectors?

The MISSION^® TRC shRNAs are cloned into the pLKO.1 transfer vector that is compatible with standard 2 plasmid (packaging vector with rev gene and envelope vector) or 3 plasmid (packaging vector without rev gene, envelope vector, and rev expression vector) packaging systems.

What restriction sites were used for cloning the shRNA sequences? I am interested because I would like to obtain some shRNA constructs, but would I need to transfer the sequences into a different vector backbone?

The shRNA was cloned into the AgeI and EcoRI sites. The individual hairpin sequences can be found on our website as well.

Does the MISSION^® TRC Library include a bar code that I could use for microarray analysis?

Currently, our vectors do not contain an internal barcode nor do we pool the clones for a gene set. You may use the individual shRNA sequence to screen resulting clonal populations for stable integration.

Can I search and design my own shRNAs on the TRC “Search for shRNA hairpin sequences page”?

The algorithm used to design the MISSION^® TRC clone library is generally the same as what is found on the TRC page. However, there have been numerous improvements to the design as the library is being cloned by the Broad-TRC. The clones that you find on the public TRC page are not the most current design version. The updated TRC clones are only available to TRC members and through purchase with Sigma-Aldrich.

The protein I intend to knockdown is very stable, does the expression of the siRNA extend over a long period, where the cells are dividing?

Since the library is lentiviral based, it allows for stable integration of the shRNA into the host chromosome. Stable clones or populations may be selected via the addition of puromycin. Individual clones may be chosen and expanded. Due to the random integration of the shRNA, screening several clones allows for optimal knockdown of your gene of interest and verification of possible off-target expression patterns.

Why doesn't the non-target control vector affect mammailian gene expression?

The non-target control vector (SHC002, SHC002V) contains an shRNA insert that does not target human and mouse genes, making it useful as a negative control in experiments using the MISSION® shRNA library clones. Please refer to the product listing for hairpin sequence and vector map.

Does pLKO.1 contain WPRE?

No, WPRE (Woodchuck Response Element) is known for its ability to enhance transription of transgenes. However, it is not believed to be necessary for transcription of shRNA. In addition, some suggest that WPRE may play a role in oncogene activity and may have implications in vivo and in clinical trials.