shRNA Transfection
Lentivirus, shRNA plasmid, or siRNA?
Choosing between Lentivirus, shRNA transfer vector, or siRNA, depends on what you are seeking to accomplish. What advantage is there for you to create a stable knockdown versus a transient knockdown? If you are running your RNAi on an easy to transfect cell line, under a transient RNAi event, then siRNA is a well defined, proven and effective method. If you are working with a primary cell, neuron, or generally hard to transfect cell, then lentiviral particles for introducing shRNA is attractive. The shRNA transfer vector alone in theory should be easy to work with insofar as culturing the cell, DNA transfection, antibiotic selection, and then collect data. However the true purpose of the lentivirus transfer vector is for packaging into lenti particles.
Establishing a stable knockdown phenotype is feasible with the use of lentiviral particles. Viral particles that have a VSV-G coat protein (Santa Cruz Biotechnology Inc.) will have broad tropism. Otherwise if you are studying effects that can be measured with transient knockdown, then siRNA is a more simple and straightforward approach. siRNA is a user friendly technique since you can calculate empirical moles of duplex/# cells or total volume (molarity), with a minimal number of steps and peripheral controls toward achieving results.
shRNA transfer vectors are ~7 kb DNA constructs that can give less stability problem since it is DNA instead of RNA. However there are considerable nuances in the actual design and cloning of these vectors, in the actual establishment of antibiotic resistance toward generating a stable cell, and in controlling for the specific silencing/reproducible results. shRNA has limitations due to the nature of having such a large delivery vector producing a small hairpin substrate, and over cell passages under antibiotic selective pressure (ie puro resistance may not = Pol III shRNA cassette expression).
TECHNICAL SERVICE GUIDE: siRNA
Catalog #
Summary:
1) Optimize the transfection reagent; measure transfection efficiency of the transfection reagent with FITC-siRNA.
2) Measure knockdown in a range of cell densities ( 30-80%) within 24-72 hours
3) Measure knockdown in a range of siRNA concentrations (30-90 nM) within 24-72 hours
Providing suggestions outlined in the notes below is worth considering and may bring success.
Background Info
§ What are the experimental results?
§ Describe how gene knockdown is measured? qPCR / Western / IF
§ How was the RNA reconstituted?
NOTE: siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution. Having the correct molarity of the solution is critical.
§ Molarity of siRNA vialed: 10 uM ( uM/L )
§ Volume after reconstitution: 330 uL
§ Mass of 1 mole of siRNA: 13800 g/mol ( 21nt X 660 g/base pair)
§ Total mols per vial: 10 um/L X 330 uL = 3.3 nm
§ Total grams per vial: 3.3 nm X 13800 g/mol = 45.5 ug
§ Solution concentration: 45.5 ug/ 330 uL = 0.138 ug/uL
§ Did this same vial or other lot of siRNA work in the past?
NOTE: If the siRNA same cat# has worked in the past, and now does not work, this may suggest RNase contamination. There are ways to determine this by running 1 pmol (17 ng) siRNA in a native 2% agarose gel, however replacing the vial is a straightforward solution.
§ Describe the cell type for this experiment?
§ What transfection reagent is used for the siRNA tranfection?
NOTE: Cationic lipid based transfection reagents (ie Lipofectimine, L2000, Transit TKO, Oligofect, Dharmafect, sc-29528) are each one a unique formula. Certain cell types will respond better to certain cationic lipid (positive charge lipophilic) reagents. For this reason, measuring transfection efficiency is necessary.
§ How was transfection efficiency measured?
NOTE: The researcher may have an existing transfection reagent that works on their cells in other experiments (ie cDNA). Suggest to try the same reagent and measure transfection efficiency.
§ What time point was transfection uptake of FITC-siRNA measured?
NOTE: Measuring transfection efficiency with sc-36869 will validate that liposome-dependent siRNA entry into the cells is taking place efficiently. It is important to measure transfection efficiency 5-7 hours post transfection since this is when the optimum time point where most transfection takes place. Common methods are IF or Flow cytomtetry.
§ Adherent cell (grows on the surface of the plate): What is the cell confluency at time of transfection?
§ Suspension cell (ie leukocytes/lymphocytes, cells are suspended in the media) : How many cell count # used to seed the well?
NOTE: A hemocytometer (cell counter) is common for counting cells for seeding into multiwell plates (6, 12, 24 well); originally designed for performing blood cell counts. Cell density is an important parameter for knockdown. Optimum cell density will vary and typically falls between 30-80%. NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%), will reveal an optimal cell density where knockdown is optimal with minimal cell death. Effective confluence can range from 30-80%.
§ What nanomolar concentration(s) of siRNA are tested?
NOTE: Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%) & a range of siRNA concentrations (30, 60, 90 nM) will reveal an optimal convergence of cell density and concentration of siRNA where knockdown is optimal with minimal cytotoxicity (cell death).
§ What time points is RNAi measured?
NOTE: 48 hours post transfection is a relevant singular point. Measuring knockdown for a few time points in the 24-72 hour window may indicate the frame when RNAi is most optimal. Titrating the siRNA concentration (30-90 nM) for the cells will indicate the best amount to see an effect.
§ How is RNAi measured? Western blot - IF - qPCR - other
NOTE: For WB, titrating the antibody may reveal subtle changes in knockdown. For IF, running secondary controls may indicate nonspecific fluorescence mistaken for signal.
§ Quantitative RT-PCR, which primers were used and what type of system?
NOTE: With appropriate internal controls (GAPDH, DNA contamination control), qPCR can be very reliable in determining translation initiation arrest.
TECHNICAL SERVICE GUIDE: Lentivirus
Catalog #
Summary:
1) Determine if the VSV-G coat protein has tropism toward the target cell; measuring transduction efficiency with sc-108084 2) Measure a range of MOI (5-10+) 3) Measure knockdown within 48-72 hours after puromycin selection
Measure transduction efficiency
§ Transduction in what cell type?
§ Primary cell or Continuous/immortal cell?
NOTE: Primary cell cultures are first generation cells from a living organism and typically have less than 5 passage lifespan. Lenti is popular for primary cells since they are difficult to transfect. Continuous or immortalized cells have the ability to proliferate indefinitely in culture.
§ Is this cell type known to have tropism for VSV-G coat protein?
§ How is transduction efficiency measured for tropism to VSV-G?
§ If a copGFP expressing Lentivirus was used to measure tropism, at what time point was transduction efficiency of the copGFP Control Lentiviral Particles or other reporter measured?
NOTE: 48 hours post-transduction is the time point where puromycin selection begins. This is also a good time point to evaluate transduction efficiency by measuring copGFP fluorescence inside the cells.
§ How was the reporter gene measured? (FCM, IF, other)
Multiplicity of Infection (MOI)
X = How many cell count was transduced? NOTE: A hemocytometer is common for this step; originally designed for performing blood cell counts, contains a etched grid on a slide, count cells/square in 5-10 squares, then average out the number and extrapolate. Y = How many uL of virus was used? NOTE: MOI = X / (Y * (particles/uL)). HEK293T and other easy to transduce cells (MOI of 5-20), while neuronal cells,SHSY5Y, may require MOI of 10-50.
§ How many [ug/ml] puromycin is added at what time point post transduction?
§ How was optimum puromycin concentration determined?
NOTE: The minimum antibiotic concentration to use is the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection (normal range; 1-10 ug/ml). Add puromycin 48 hours post transduction.
§ Western blot, IF or Quantitative RT-PCR?
§ Negative controls (scrambled hairpin virus, no virus)?
NOTE: Running a parallel transduction with no virus should yield 100% cytotoxicity upon puromycin addition. Scrambled hairpin virus (sc-108080) transduction is useful to determine if any other aspect of the transduction process influences knockdown, including the presence of a non gene specific hairpin (nonspecific antisense).
Instead of chemically synthesizing the siRNAs before introducing it in the cell, the siRNAs are made directly by the cells through an expression vector that is transiently transfected into a dividng cell. The shRNA transfer vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery. While transient transfection is advantageous for fast analysis of shRNA mediated effects, stable transfection ensures long-term, reproducible as well as defined shRNA effects.
For many disease models, the most desirable cell types such as immune system or primary cells are not amenable to transfection. Viral delivery of RNAi vectors is a powerful alternative to transfection for these cell types as well as for in vivo applications. Stable expression is achieved by integration of the gene of interest into the target cell's chromosome: Initially the shRNA of interest has to be introduced into the cell, subsequently into the nucleus, and finally it has to be integrated into chromosomal DNA.
Stable expression can be influenced by two factors: The transfection method used and the vector containing the shRNA of interest. The transfection method determines which cell type can be targeted for stable integration through antibiotic selection. While many lipofection reagents transfect DNA up to a certain amount into adherent cell lines, efficient delivery of DNA into difficult-to-transfect suspension cell lines or even primary cells is only possible with viral methods and nucleofection.
Nucleofection is a non-viral method of introducing DNA molecules into the nucleus of dividing cells, therefore significantly increasing the chances of chromosomal integration of the transgene. The technology is pioneered by Amaxa
Santa Cruz Biotechnology, Inc. does not disclose vector map information for the sh plasmids, including RE. This removes variable of a single cut RE in the empty showing up in a cloned-in sh,
The tech writing for Lonza nucleofection would suggest a linear plasmid has more efficient outcome for GFP expression , however
§ Values are so close between linear and circular DNA for the 2 and 5ug event looking at GFP expression and there is no claim that linear is necessary.
§ n= 2 cell lines tested provides limited insight into the validity of the claim for such a device as this : http://www.lonzabio.com/technology.html
§ Transgene study only - there is no mention of RNAi or lenti/sh vector suitability to this tech in their nucleofection literature -
Although there is still some debate as to the effectiveness of this approach, a regular shRNA transfer vector may be able to integrate into the genome of the target cell by antibiotic selection alone. The process may occur randomly by the cell's machinery itself, possibly via DNA repair and recombination enzymes. If this phenomenon does occur, integration into inactive heterochromatin may result in little or no shRNA expression, whereas integration into active euchromatin may allow for shRNA expression. However, random integration could also lead to silencing of the shRNA cassette. Several strategies have been developed to overcome the negative position effects of random integration: Site-specific, homologous and transposon-mediated integration strategies are used but require the expression of integration enzymes or additional sequences on the plasmid.
Lentiviral particles are highly efficient at infection and stable integration of the shRNA into a cell system. To obtain the lentiviral particle, the transfer vector that contains the shRNA cassette is already flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the shRNA cassette into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the shRNA into pseudovirus particles. Viral proteins which make virus shells are provided in the packaging cell line (HEK 293T), but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient. Lentiviral particles can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell.
§ Lentiviral systems efficiently transduce both dividing and non-dividing cells
§ Study long-term gene knockdown with stable expression
§ Reproducibly transduce cell populations
§ Inducible or constitutive gene knockdown
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