DNA Vector-based RNA Interference to Study Gene Function in Cancer

Summary

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

Abstract

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 possible^2,3. 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 cancer^4,5, 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.

Protocol

1. Generation of shRNA Constructs

1. Identify a siRNA-target sequence (20-23 nucleotides) based on previously published criteria⁶ or use a web-based algorithm server, such as "siRNA Target Finder" from Ambion and GenScript.
2. Synthesize oligonucleotides based on the design of Figure 1 and example sequences in Table 1. Carry out PCR amplification using the oligonucleotides P1 and P2 (30 cycles of denaturing at 94 °C for 30 sec, annealing at 60 °C for 30 sec, and elongating at 72 °C for 30 sec) and a plasmid carrying the U6 or H1 promoter as a template. Purify the PCR product using a PCR purification column. ....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
PCR thermocycler
Ultracentrifuge
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
Materials List