Who can access the services of the Einstein shRNA Core Facility?
Full services are available to researchers from Albert Einstein College of Medicine, Montefiore Medical Center, and Yeshiva University. Screening services and limited distribution of shRNAs as lentivirus are also available to researchers at non-profit entitities that are members of the New York Structural Biology Center Consortium (Memorial Sloan Kettering Cancer Center, Columbia University, Rockefeller University, New York University, Mt. Sinai School of Medicine, State University of New York, City University of New York, Weill Cornell Medical Center, and the Wadsworth Center of the New York State Department of Health). For more information, contact us at shRNA@einstein.yu.edu.
How can I find out what shRNA clones are available for my gene of interest?
Send the gene symbol (e.g. GAPDH), the gene ID (e.g. 2597), and the species of interest to shRNA@einstein.yu.edu. (Gene symbol and gene ID can be looked up using the NCBI Gene resource.) We will send you a report listing clones available, along with their sequences and target sites.
Which library should I order clones from?
The TRC and GIPZ libraries differ in their shRNA design strategy and in the vectors used for library construction. As a result, one may be more suitable than the other for a particular application. The TRC library uses the pLKO.1 vector, which expresses simple hairpin structures from a Pol III U6 promoter. The GIPZ library is built on the pGIPZ vector, which uses a Pol II CMV promoter to drive expression of shRNA sequences that are incorporated into the mir30 sequence context. The pGIPZ vector encodes a GFP marker, making it possible to monitor transduced cells directly. However, the marker precludes use of this vector in cell systems that already use a GFP-based reporter. The choice of promoter may be important in some cases as well. The CMV promoter is subject to silencing in some cell types.
How many clones should I order for each gene?
Since not all shRNAs silence their targets with equal efficiency, it is important to test several for each gene to find an effective one. Moreover, conclusions drawn from knockdown experiments are much stronger if the same cellular phenotype can be independently produced using multiple shRNAs targeting the same gene. We suggest testing at least four independent shRNA sequences, and we offer a multi-clone discount when ordering four or more shRNAs from the same library targeting a single gene.
What format should I order?
In choosing which format to order, keep in mind that our customers report a 40% success rate when starting from glycerol stock or plasmid, and a 72% success rate when the shRNA Core Facility generates the lentivirus for them. The higher success rate on a per-shRNA basis reflects our standardized procedures and experience in packaging and titering lentivirus. If you are adept at making lentiviral preps, we suggest you save money by ordering glycerol stocks. However, if you are not familiar with the procedures, you may find it more advantageous and cost-effective to order virus from us.
Should I use shRNA or CRISPR/Cas9 to down-modulate gene expression?
There are potential advantages and disadvantages to each approach. CRISPR/Cas9 produces a permanent change at the DNA level; shRNA leads to mRNA degradation and requires continual expression of the shRNA to maintain the effect. CRISPR causes a complete knockout of gene function; shRNA reduces expression signficantly but usually leaves some residual gene expression. Residual expression can be desirable if complete knockout is lethal, or undesirable if the phenotype of interest requires complete loss of function. Both methods are subject to potential off-target effects, but early indications are that these are less of a concern with CRISPR. Multiplexing is limited with shRNA but possible with CRISPR. CRIPSR is more versatile, with modifications posssible that allow it to be used for mutagenesis, activation, repression, and other applications; shRNA is useful for gene down-regulation only. Effects of shRNA can usually be seen fairly quickly following transduction; CRISPR-based knockout may require several days of Cas9 expression to manifest in most of the cell population.
How do I know if my cells are transducible?
Most cell types are readily transducible by lentivirus. If you are uncertain about your cells, contact us and we can supply you with a small quantity of control virus expressing GFP for testing transducibility.
How can I avoid off-target effects?
shRNAs, like siRNAs, can exhibit off-target effects, in which genes other than the intended target are silenced. These effects usually arise, through a miRNA-like mechanism, from sequence complementarity between the ~6-base "seed" region at the 5' end of one of the siRNA strands and a target site in the 3' UTR of a transcript. Off-target effects are difficult to predict and should always be kept in mind as a possible source of phenotypic effects. In practice, it is best to take these experimental steps to reduce the impact of off-target silencing: (1) test several shRNAs and identify at least two independent sequences that produce the same phenotype; and (2) show that these shRNAs produce knockdown at the protein (e.g. by Western blot) or mRNA (e.g. by branched DNA or qRT-PCR) level.
What controls are available?
We offer the following controls:
- TRC libraries
- Empty pLKO.1 vector (no shRNA insert)
- pLKO.1 clone targeting eGFP (can function as positive control or as non-targetin control)
- GIPZ libraries
- Empty pGIPZ vector (no shRNA insert)
- Non-silencing control (shRNA designed to not target any mammalian gene)
- GAPDH shRNA (positive control)
- LentiORF libraries
- pLOC clone encoding RFP (in addition to the GFP reporter present on all pLOC clones; for Precision LentiORF library)
- Emtpy pLX 304 vector (for hORFeome V8.1 library)