Griffin:shRNA Transfection

shRNA Transfer Vector Overview

Short hairpin RNAs (shRNAs) are typically modeled after miRNA hairpin precursors and cloned into a transfer vector that is suitable for either direct transient transfection of the shRNA, or for packaging into a lentiviral particle.

Transient shRNA Transfection

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.

Stable shRNA (Lentiviral) Transfection

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 Particle Overview

Lentiviral vectors are usually created in a transient transfection system in which a cell line (HEK293T) is co-transfected with three separate plasmid expression systems. These include the transfer vector plasmid (portions of the HIV provirus & shRNA cassette), the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (ENV) of a different virus. The three plasmid components of the vector are put into a packaging cell which is then inserted into the HIV shell. Pseudoviral particles are then collected and must undergo titration to measure infectivity prior to experimental use.

Transfer Vector Plasmid

The transfer vector plasmid contains cis-acting genetic sequences necessary for the vector to infect the target cell and for transfer of the shRNA cassette, and contains restriction sites for insertion of the shRNA. The 3’ and 5’ LTRs, the original envelop proteins, and gag sequence promoter have been removed.

Envelop Gene Plasmid

The third plasmid’s envelope gene of a different virus specifies what type of cell to target and infect. Normally HIV can infect only helper T-cells because they use their gp120 protein to bind to the CD4 receptor. However, it is possible to genetically exchange the CD4 receptor-binding protein for another envelope protein that codes for different cell types. This gives the HIV lentiviral vector a broad range of possible target cells. There a few types of heterologous envelope proteins. Envelopes include the ecotropic envelope, amphotropic envelope, gibbon ape leukemia virus (GALV) envelope, feline endogenous virus envelope (RD114), and the (G glycoproteins of the vesicular stomatitis virus) VSV-G envelope.

There are two major points to consider when choosing which coat (pseudotype) to use in the laboratory. First, because VSV/G pseudotyped virus may infect human tissue, it may not be preferable to use if one is working strictly in a mouse system. Working with human infectious agents will likely require a heightened BLS lab safety level. A second point of consideration is the limitations imposed by the ecotropic coat: it is difficult to concentrate the virus due to the inherent instability of the ecotropic coat. Virus ultracentrifugation concentration is often necessary when infecting certain cell types or when generating transgenic mice, and for this reason, many choose to use VSV/G pseudotyped virus because the particles are stable to the high g-forces of ultracentrifugation. Even with highly concentrated virus, there is a possibility that the VSV/G or ecotropic pseudotypes may still display differing cell-type specificities for infection, an important consideration when planning an experiment. For example, in the CNS, VSV/G mediates vector entry primarily in neurons, while astrocytes appear to be better infected using other envelopes.

Packaging Plasmid

The packaging plasmid is the backbone of the virus system. In this plasmid are found the elements required for vector packaging such as structural proteins, HIV genes (except the gene env which codes for infection of T cells, or the vector would only be able to infect these cells), and the enzymes that generate vector particles. Also contained is the human cytomegalovirus (hCMV) which is responsible for the expression of the virus proteins during translation. The packaging signals and their adjacent signals are removed so the parts responsible for packaging the viral DNA have been separated from the parts that activate them. Thus, the packaging sequences will not be incorporated into the viral genome and the virus will not reproduce after it has infected the host cell.

All Medline abstracts: PubMed | HubMed

Vector Titration

Vector titration typically involves PCR or flow cytometry depending on the presence of a fluorescent protein. For example, mouse NIH 3T3 or human HT1080 cells are infected for 4 hours with limiting dilutions of vector supernate in the presence of polybrene and analyzed for expression of fluorescent protein or detection of vector or packaging sequences by PCR. The titer represents the relative infectivity of the vector as measured on the target cell of choice and is expressed as Infectious Units (IU)/mL. Different target cells, or different infection protocols, may yield different results.

Vector supernate can be concentrated using ultrafiltration. Recoveries (65-90%) vary depending on the envelope used and stability of the viral particle. This technique does not separate empty from functional viral particles. Therefore, at very low titers, concentration may not always yield an improved titer due to the presence of inhibitory empty viral particles.

Packaging cells

Human 293 cells are usually used for packaging, since they can be transfected with efficiencies in the range of 90-100%. If the cells do not transfect well in control experiments using a non-viral GFP vector, it may be worth discarding them and trying a different lot of cells. On occasion, cells can become refractive to transfection. Consequently, use low passage cells for all transfections. Although 293 cells are adherent, they can easily detach from the plate during pipetting, or even by jostling a plate that has reached confluency. For this reason, perform the transfection when the cells are approximately 30% confluent. After 36-48 hours, the cells should reach confluency and be producing the maximum amount of virus. If the cells are fed fresh media, several harvests of virus can be made after the cells reach confluency. One final note: if human cells are used for packaging a RNA hairpin that targets a human sequence, lower viral titers may be experienced if silencing of the targeted gene is detrimental to the cell or packaging process.

shRNA Transfer Vector Promoters

RNA Polymerase III type 3 promoters are common in vector-based expression of shRNAs. The most abundant cellular RNAs transcribed from a type III RNA pol III promoter are the U6 small nuclear RNA (which play a crucial role in the processing of premature RNA); the 7SK RNA (a negative regulator of RNA polymerase II elongation factor TEFb); and H1 RNA (a component of RNAse P).

Unlike the majority of RNA pol III promoters, type 3 RNA Pol III promoters do not require any additional sequence elements. In addition, termination of transcription by Pol III occurs at definitive tracts of 4–5 thymidines (T4–5), which can be inserted downstream of shRNA coding sequences in order to ensure direct termination. These 2 specific properties enable the U6, 7SK, and H1 promoters to be ideal candidates for in vivo delivery systems of siRNAs where DNA templates can undergo transcription into small RNAs with structural features resembling active siRNA/miRNA.

U6

U6 small nuclear RNA (snRNA) is an essential component of the eukaryotic spliceosomes, is unique in that it is synthesized by RNA polymerase III, while all other U-snRNAs are synthesized by RNA polymerase II. U6 genes are notable for functional upstream regulatory elements which resemble RNA polymerase II regulatory sequence motifs. Tests for both potency and adverse metabolic effects upon primary cells indicate that U6 promoter may exert toxicity relative to H1 or 7SK due in part to higher activity.

7SK

7SK is an abundant and evolutionarily conserved small nuclear RNA discovered in the 1970s. 7SK is transcribed by RNA polymerase III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (Murphy et al 1984)]. The human 7SK promoter presents a strong permissivity for the nucleotide in the +1 position and recognizes a cluster of 4 or more T residues as a termination signal. The human 7SK promoter is ideal for the production of shRNAs as it can generate high amounts of shRNAs (Czauderna et al 2003, Koper-Emde et al 2004). In a series of experiments aimed to compare the strength of the human 7SK, H1 and U6 promoters, the best silencing efficiencies of various target genes was consistently obtained with a 7SK promoter (unpublished data;Invivogen).

H1

The H1 promoter drives expression of a unique gene encoding H1 RNA, the RNA component of the human RNase P. The H1 RNA gene is transcribed by RNA polymerase III into a small RNA transcript. The human H1 promoter presents the characteristics of being unusually compact. All the essential elements for transcription, that is octamer, Staf, proximal sequence element and TATA motifs, lie within 100 bp of the 5‘ flanking region. This promoter is highly permissive for the nucleotide at the +1 position which is originally an A. The termination sequence consists of a stretch of 5 thymidines. The cleavage of the transcript after the termination signal occurs after the second uridine generating a 2 nt 3‘ overhang. The H1 promoter has been successfully used in different plasmids to create siRNAs.

All Medline abstracts: PubMed | HubMed

shRNA Transfer Vector Transfection Reagents

Gene silencing using RNAi is critically dependent on highly efficient delivery of shRNA or siRNAs into cells. The two conventional reagent types are Cationic lipid-based and Polymeric formulations. All commercial transfection reagents are proprietary formulations that are competing for market share by claiming certain advantages (ie, broad cell type compatibility, low cytotoxicity, high efficiency).

Cationic lipid

Cationic lipid transfection reagents are suitable for transfecting into a wide variety of dividing cell cultures. Commercial examples include: Lipofectamine / L2000, Dharmafect, iFect, and TransIT TKO. Cationic lipids work by forming lipsomal vesicles that house the siRNA payload and bleb their way through the living cell membrane and into the cytoplasm. The efficiency of this process must be determined in order to have confidence in the knockdown effects. There are numerous commercial sources for transfection reagents for good reason; there are numerous cell types and lipsome structure will influence transfection efficiency in the multitude of experimental cell types that exist.

Polymeric

Polymeric formulations have been developed and optimized for transfection of shRNA plasmid DNA into the nucleus of cultured eukaryotic cells by vendors such as Open Biosystems. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus (Pollard et al 1998). Polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

shRNA Transfer Vector Transient Transfection Procedure

In a six well tissue culture plate, grow cells to a 50-70% confluency in antibiotic-free normal growth medium supplemented with FBS.

NOTE: This protocol is recommended for a well from a 6 well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.

NOTE: Healthy and subconfluent cells are required for successful transfection experiments. It is recommended to ensure cell viability one day prior to transfection.

Prepare the following solutions:

NOTE: The optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio should be determined experimentally beginning with 1 μg of shRNA Plasmid DNA and between 1.0 and 6.0 μl of shRNA Plasmid Transfection Reagent as outlined below. Once the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio has been identified for a given cell type, the appropriate amount of shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency. For example, if the optimal shRNA Plasmid DNA:shRNA Plasmid Transfection Reagent ratio is 1 μg:1 μl, then amounts ranging from 0.5 μg/0.5 μl to 2.0 μg/2.0 μl should be tested.

Solution A: For each transfection, dilute 10 μl of resuspended shRNA Plasmid DNA (i.e. 1 μg shRNA Plasmid DNA) into 90 μl shRNA Plasmid Transfection Medium (serum antibiotic free medium).

Solution B: For each transfection, dilute 1 - 6 μl of shRNA Plasmid Transfection Reagent with enough shRNA Plasmid Transfection Medium to bring final volume to 100 μl.

NOTE: Do not add antibiotics to the shRNA Plasmid Transfection Medium.

NOTE: Optimal results may be achieved by using siliconized microcentrifuge tubes.

NOTE: Although highly efficient in a variety of cell lines, not all shRNA Plasmid Transfection Reagents may be suitable for use with all cell lines.

Add the shRNA Plasmid DNA solution (Solution A) directly to the dilute shRNA Plasmid Transfection Reagent (Solution B) using a pipette. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature.

Wash the cells twice with 2 ml of shRNA Transfection Medium. Aspirate the medium and proceed immediately to the next step. NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency.

NOTE: Do not use PBS as the residual phosphate may compete with DNA and bind the shRNA Plasmid Transfection Reagent, thereby reducing the transfection efficiency. For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.

For each transfection, add 0.8 ml shRNA Plasmid Transfection Medium to well.

Add the 200 μl shRNA Plasmid DNA/shRNA Plasmid Transfection Reagent Complex (Solution A + Solution B) dropwise to well, covering the entire layer.

Gently mix by swirling the plate to ensure that the entire cell layer is immersed in solution.

Incubate the cells 5-7 hours at 37° C in a CO2 incubator or under conditions normally used to culture the cells.

NOTE: Longer transfection times may be desirable depending on the cell line.

Following incubation, add 1 ml of normal growth medium containing 2 times the normal serum and antibiotics concentration (2x normal growth medium).

Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.

Aspirate the medium and replace with fresh 1x normal growth medium.

Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the step above.

NOTE: Controls should always be included in shRNA experiments. Control shRNAs are available as 20 μg. Each encode a scrambled shRNA sequence that will not lead to the specific degradation of any known cellular mRNA.

NOTE: For Western blot analysis prepare cell lysate as follows: Wash cells once with PBS. Lyse cells in 300 μl 1x Electrophoresis Sample Buffer (sc-24945) by gently rocking the 6 well plate or by pipetting up and down. Sonicate the lysate on ice if necessary.

NOTE: For RT-PCR analysis isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially available RNA isolation kit.

shRNA Controls

Negative Control: Untreated Cells. Untreated cells will provide a reference point for comparing all other samples.

Negative Control: Empty construct, containing no shRNA insert; The empty viral particles or DNA are a useful negative control that will not activate the RNAi pathway because it does not contain an shRNA insert. It will allow for observation of cellular effects of the transduction/transfection process. Cells transduced/transfected with the empty control provide a useful reference point for comparing specific knockdown.

Negative Control: Non-targeting shRNA; This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence cotnains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transduction/transfection on gene expression. Cells transduced/transfected with the non-target shRNA will also provide useful reference for interpretation of knockdown.

Positive Control: Positive reporter vector or lentiviral particles; This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The GFP Control contains a gene encoding GFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection.

Positive Control: Positive shRNA knockdown control; This control contains shRNA sequence that targets GFP expression. This shRNA control has been experimentally shown to reduce GFP expression. This control serves to quickly visualize knockdown in cells expressing GFP.

Positive Control: Positive shRNA knockdown control; This control contains shRNA sequence that targets eGFP expression (GenBank Accession # pEGFP U476561). The shRNA has been experimentally shown to reduce eGFP expression by 90% in C166-GFP mouse fibroblast cells 48 hours post-transduction by mRNA transcript level. This control serves to quickly visualize knockdown in cells expressing eGFP.

Factors Influencing Successful Transfection

1. Concentration and purity of nucleic acids – Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.

2. Transfection in serum-free media – the highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.

3. No antibiotics in transfection medium – The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.

4. High protein expression levels – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.

5. Cell history, density, and passage number–It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.

References

All Medline abstracts: PubMed | HubMed

Griffin:shRNA Transfection

Contents

shRNA Transfer Vector Overview

Transient shRNA Transfection

Stable shRNA (Lentiviral) Transfection

Lentiviral Particle Overview

Transfer Vector Plasmid

Envelop Gene Plasmid

Packaging Plasmid

Vector Titration

Packaging cells

shRNA Transfer Vector Promoters

U6

7SK

H1

shRNA Transfer Vector Transfection Reagents

Cationic lipid

Polymeric

shRNA Transfer Vector Transient Transfection Procedure

shRNA Controls

Factors Influencing Successful Transfection

References

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