Griffin:shRNA Transfection

shRNA Overview

Short hairpin RNAs (shRNAs) are typically modeled after miRNA hairpin precursors and cloned into expression vectors. In this system 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 can either be transiently transfected into a dividng cell, or packaged into a lentiviral particle for stable transfection. The shRNA expression vector alone can be transiently introduced into the dividing cell where the shRNA is synthesized by cellular machinery.

To obtain the lentiviral particle, the shRNA containing vector sequence that is flanked by LTRs and the Psi-sequence of HIV requires an additional few steps. 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 in virions. 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 retrovirus 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.

shRNA 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.

1. An DS, Qin FX, Auyeung VC, Mao SH, Kung SK, Baltimore D, and Chen IS. Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol Ther. 2006 Oct;14(4):494-504. DOI:10.1016/j.ymthe.2006.05.015 |
2. Myslinski E, Amé JC, Krol A, and Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acids Res. 2001 Jun 15;29(12):2502-9. DOI:10.1093/nar/29.12.2502 |
3. Tiscornia G, Singer O, Ikawa M, and Verma IM. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1844-8. DOI:10.1073/pnas.0437912100 |
4. Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC, and Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5515-20. DOI:10.1073/pnas.082117599 |
5. Danzeiser DA, Urso O, and Kunkel GR. Functional characterization of elements in a human U6 small nuclear RNA gene distal control region. Mol Cell Biol. 1993 Aug;13(8):4670-8. DOI:10.1128/mcb.13.8.4670-4678.1993 |
6. Koper-Emde D, Herrmann L, Sandrock B, and Benecke BJ. RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter. Biol Chem. 2004 Sep;385(9):791-4. DOI:10.1515/BC.2004.103 |
7. Yu JY, DeRuiter SL, and Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6047-52. DOI:10.1073/pnas.092143499 |

shRNA 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 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

1. Murphy S, Altruda F, Ullu E, Tripodi M, Silengo L, and Melli M. DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome. J Mol Biol. 1984 Aug 25;177(4):575-90. DOI:10.1016/0022-2836(84)90038-x |
2. Czauderna F, Santel A, Hinz M, Fechtner M, Durieux B, Fisch G, Leenders F, Arnold W, Giese K, Klippel A, and Kaufmann J. Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res. 2003 Nov 1;31(21):e127. DOI:10.1093/nar/gng127 |
3. Koper-Emde D, Herrmann L, Sandrock B, and Benecke BJ. RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter. Biol Chem. 2004 Sep;385(9):791-4. DOI:10.1515/BC.2004.103 |
4. Whither RNAi?. Nat Cell Biol. 2003 Jun;5(6):489-90. DOI:10.1038/ncb0603-490 |
5. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, and Escande D. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J Biol Chem. 1998 Mar 27;273(13):7507-11. DOI:10.1074/jbc.273.13.7507 |

• Open Biosystems
• Invivogen
• Santa Cruz Biotechnology, Inc.
• System Biosciences