Векторной ДНК на основе РНК-интерференции по изучению функции генов в Раке
Summary
РНК-интерференция (RNAi) обладает многими преимуществами по сравнению с геном нокаут и был широко использован в качестве инструмента в геном функциональные исследования. Изобретение векторной ДНК на основе РНК-интерференции технологии сделали долгосрочный и индуцибельной гена нокдаун возможно, а также увеличить возможности генов<em> В естественных условиях</em>.
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 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.
Protocol
Discussion
Этот протокол описывает метод, чтобы сбить генов использованием ShRNA и визуализировать его биологический эффект использования биолюминесцентного визуализация роста опухолевых клеток в живом организме. Целевой сайт ShRNA не должны содержать любой из трех сайтов рестрикции (BamHI, HindIII …
Declarações
The authors have nothing to disclose.
Acknowledgements
Эта работа была частично поддержана исследований ученый грантов (116403-RSG-09-082-01-ГГО) из Американского онкологического общества и очный средств Wake Forest University Health Sciences для GS. DBS была поддержана NCI обучение грант 5T32CA079448.
Materials
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.
| Name of the reagent |
| 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
Referências
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