1Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, 2Department of Pathology and Comprehensive Cancer Center, Wake Forest University School of Medicine
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Stovall, D. B., Wan, M., Zhang, Q., Dubey, P., Sui, G. DNA Vector-based RNA Interference to Study Gene Function in Cancer. J. Vis. Exp. (64), e4129, doi:10.3791/4129 (2012).
RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression.
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model.
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.
1. Generation of shRNA Constructs
2. Lentivirus Transfection
Precautions: Lentiviral vectors are derived from the human immunodeficiency virus-1 (HIV-1) genome and replication incompetent. They have been widely used in gene delivery due to the capability of infecting both dividing and non-dividing cells. However, two major risks exist in the studies using lentivirus. (1) The potential for generation of replication-competent lentivirus. This risk can be greatly reduced if the third generation lentiviral system is used. (2) The potential for oncogenesis. This risk can be exacerbated if the carried inserts are oncogenic or repress tumor suppressors. Research activities involved in HIV-based lentivirus should follow the "Biosafety Considerations for Research with Lentiviral Vectors" of the NIH (http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html) and require an approval from the Biosafety Committee of a researcher's institution. Generally, enhanced Biosafety level-2 (BSL-2) containment is required for the laboratory setting if lentiviral vectors are used.
3. Infection and Characterization of Infected Cells
4. Xenograft Mouse Model Study
5. Representative Results
This protocol was used to study the effects of YY1 knockdown on xenograft tumor formation of Firefly luciferase- expressing MDA-MB-231 cells (human breast adenocarcinoma cells; Caliper Life Sciences) in athymic nude mice. The shRNA target sequence of human YY1 is "GGG AGC AGA AGC AGG TGC AGA T". A scrambled sequence "GGG ACT ACT CTA TTA CGT CAT T" was also created for a control (cont) shRNA, which did not have significant similarity to any known transcript. The oligonucleotides used to make Tet-On inducible shRNA constructs, with YY1 as an example, are shown in Table 1. As a result, two lentiviral vectors, pLu-Puro-Indu-YY1 shRNA and pLu-Puro-Indu-cont shRNA, were constructed and they were used to produce lentiviruses. We used these lentiviruses to individually infect two MDA-MB-231 cell clones (clones 1 and 3) expressing both tetracycline regulator (tetR) and Firefly luciferase. Polyclonal cell populations were obtained after puromycin selection. Figure 3A shows Dox-induced YY1 knockdown in these MDA-MB-231 cells infected by pLu-Puro-Indu-YY1 shRNA lentivirus. We then used the polyclonal cells of clone 3 infected individually by these inducible YY1 shRNA and cont shRNA lentiviruses and observed that YY1 depletion reduced invasiveness of MDA-MB-231 cells (Figure 3B). Western blot analyses confirmed Dox-induced YY1 silencing in MDA-MB-231 cells with the indu-YY1 shRNA, while the cells containing indu-cont shRNA did not show this effect (Figure 3C). We then used these cells for the xenograft mouse model study. Compared to the control groups, the mice implanted by the MDA-MB-231 cells with indu-YY1 shRNA and supplied with Dox-containing water showed reduced tumor formation, when visualized by bioluminescence (Figure 4A) and determined by tumor weights (Figure 4B). YY1 silencing in these xenograft tumors was confirmed by Western blot studies shown in Figure 4C.
Figure 1. Schematic diagram for the generation of a shRNA construct and shRNA transcription. The primers P1 to P6 are shown (see Table 1 for example sequences). Pol III: RNA polymerase III. (d): digested end. Drawing is not to scale. Click here to view larger image.
Figure 2. Schematic diagrams of (A) a lentiviral vector and (B) the mechanism of the Tet-On inducible H1 promoter used for gene silencing. The lentiviral vector was reconstructed based on pLL3.714 . H1-Pr: H1 promoter; Ubc-Pr: Human ubiquitin C promoter; Puro: puromycin; TRE: Tetracycline responsive element; Dox: doxycycline. TetR: tetracycline regulator. 5'LTR, 3'SIN-LTR and WRE are essential components of a lentiviral vector14 .
Figure 3. Dox-induced YY1 silencing and its effect on invasiveness of MDA-MB-231 cells. A. YY1 levels in two polyclonal cell populations derived from TetR-expressing clones 1 and 3 infected by pLu-Puro-Indu-YY1 shRNA lentivirus and cultured in the absence and presence of Dox. B. Boyden chamber assay of MDA-MB-231 cells with inducible shRNAs. (* P < 0.05 versus other three groups). C. Representative Western blots of YY1 expression in these four cell populations.
Figure 4. Effects of induced YY1 silencing on xenograft tumor formation by MDA-MB-231 cells. A. Schematic diagram of cell implantation (left) and representative bioluminescent images captured by the IVIS Imaging System at 4 weeks (right) post cell inoculation. B. Xenograft tumor weights at 4 weeks. * P ≤ 0.05. C. Western blots of YY1 and β-actin expression in xenografts of MDA-MB-231 cells with indu-YY1 shRNA in the absence and presence of Dox. Labels on the top are the names of individual mice.
|Oligonucleotide||Sequence (5' - 3')||Usage and location|
|P1 (Tet-On H1)||cagt GGATCC CGA ACG CTG ACG TCA TCA ACC C||PCR; at 5'-end of the H1 promoter|
|P1 (U6)||cagt GGATCC GAC GCC GCC ATC TCT AGG||PCR; at 5'-end of the U6 promoter|
|P2 (for YY1 with Tet-On H1)||cagc AAGCTT GAA atc tgc acc tgc ttc tgc tcc c GGG ATC TCT ATC ACT GAT AGG GAA C||PCR; at 3'-end of the Tet-On inducible H1 promoter|
|P2 (for YY1 with U6)||cagc AAGCTT GAA atc tgc acc tgc ttc tgc tcc c aaa caa ggc ttt tct cca agg gat a||PCR; at 3'-end of the U6 promoter|
|P3 (for YY1)||agc tt atc tgc acc tgc ttc tgc tcc c ttttt g||To be annealed with P4|
|P4 (for YY1)||aattc aaaaa ggg agc aga agc agg tgc aga t a||To be annealed with P3|
|P5 (for pLL3.7)||ggg tac agt gca ggg gaa aga ata g||For PCR screening, used with P6|
|P6 (for Tet-On H1)||GAT TTC CCA GAA CAC ATA GCG AC||For PCR screening, used with P5|
|P6 (for U6)||AGG GTG AGT TTC CTT TTG TGC TG||For PCR screening, used with P5|
Table 1. Synthesized oligonucleotides used in generating shRNA constructs. P1 and P6 sequences for the mouse U6 and Tet-On inducible human H1 promoters are provided. Human YY1 is used as an example with a shRNA target sequence of GGG AGC AGA AGC AGG TGC AGA T. The sequences specific to YY1 are highlighted. Two P2 sequences of YY1 are designed for the two promoters, respectively. The constitutive U6/YY1 shRNA was described previously15, while the Tet-On H1/YY1 was used in this protocol. P5 is vector-specific and the sequence for pLL3.714 is shown. The sequences of restriction enzymes are underlined, while the sequences to anneal to the promoters during PCR amplification are italicized.
This protocol describes a method to knock down a gene using shRNA and visualize its biological effect using bioluminescent imaging of tumor cell growth in vivo. The target site of a shRNA should not contain any of the three restriction sites (BamHI, HindIII and EcoRI) used for subcloning. In a rare scenario when any of these sites is present, an additional restriction enzyme can be used to replace it in generating the construct.
Several key steps in this protocol will speed up the construction of shRNA lentiviral vectors. First, the PCR template plasmid containing the U6 or H1 promoter may have a different antibiotic resistance gene (such as kanamycin) from the lentiviral vector (typically ampicillin). This can reduce the background of transformation caused by the template plasmid. Second, it is necessary to use competent E. coli cells with high efficiencies of transformation. A protocol using potassium and manganese ions can be used to produce competent E. coli cells with extremely high competencies12. Third, a selectable marker can facilitate the 3-fragment subcloning. For example, a lentiviral vector can be engineered to contain an expression cassette for β-galactosidase (LacZ) that will be replaced by the insertion of the PCR fragment and annealed P3/P4 oligonucleotides. In this case, recombinant plasmids with the inserts will form white colonies, while these without the inserts will be blue, when exposed to X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside).
The quality of lentiviral vectors and the three packaging plasmids is essential to efficient lentivirus production. A very economic and efficient transfection method using calcium phosphate precipitation has been provided. Polyethylenimine13 or most commercial transfection reagents can also be used in lentivirus production. To use a proper MOI for infection, it is important to determine the titers of produced lentivirus.
Mice of all experimental groups should be housed in the same room to eliminate their circadian rhythm difference that may cause variation of bioluminescence during xenograft tumor imaging. Approaches of mouse euthanasia include CO2 asphyxiation and overdose by isofluorane and should be approved by the institutional ACUC.
No conflicts of interest declared.
This work was supported in part by the Research Scholar Grants (116403-RSG-09-082-01-MGO) from the American Cancer Society and intramural funds of Wake Forest University Health Sciences to GS. DBS was supported by NCI training grant 5T32CA079448.
|In addition to common laboratory equipment used for RNA and protein analyses and cell culture, the following materials and reagents are required for this protocol.|
|Biosafety Cabinet suitable for Biosafety Level 2 containment|
|Anesthesia-induction chamber with isoflurane scavengers|
|Digital Vernier caliper|
|IVIS imaging system|
|Highly competent DH5a E. coli|
|EcoRI, BamHI, & HindIII restriction enzymes|
|T4 DNA Ligase|
|Third generation lentivirus packaging plasmids VSV-G, pRSV-Rev and pMDLg/pRRE|