Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts
逆转录病毒介导小鼠胚胎成纤维细胞的恶性转化
发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.844 浏览次数: 11076
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The authors used this protocol in:
Mar 2013
Abstract
Cellular transformation is a widely used method to artificially induce cells to form tumours in vivo. Here, we describe the methodology for malignant transformation of mouse embryonic fibroblasts (MEFs) for transplantation into immunodeficient nude mice, as used in Leong et al. (2013). The two-step process involves: 1) down-regulation of Trp53 expression using a short hairpin RNA (shRNA); and 2) overexpression of the oncogenic HRasV12 protein. Reduction of Trp53 expression leads to cell immortalisation, and the subsequent overexpression of oncogenic HRasV12 results in malignant transformation of a cell.
Keywords: Transformation (转型)
Mouse embryonic fibroblasts (小鼠胚胎成纤维细胞)
HRasv12 (hrasv12)
P53 knockdown (p53基因敲除)
Materials and Reagents
- Source of tissue: body of embryonic day 13.5 mouse embryos, harvested fresh from pregnant females
- Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 )
- Fetal Calf Serum (FCS) (Life Technologies, Gibco®, catalog number: 10437-028 )
- Trypsin (Life Technologies, Gibco®, catalog number: 25200056 )
- Dulbecco’s Phosphate Buffered Saline (PBS), without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14190-144 )
- Retroviral supernatant containing LMP-p53.1224 shRNA construct (Dickins et al., 2005)
- Retroviral supernatant containing pWZL-HRasV12 cDNA construct (Serrano et al., 1997)
- Hygromycin B (Life Technologies, catalog number: 10687-010 )
- Puromycin (Sigma-Aldrich, catalog number: P9620-10ML )
- Hexadimethrine bromide/Polybrene (Sigma-Aldrich, catalog number: H9268 )
- Polybrene (1,000x stock) (see Recipes)
Equipment
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Leong, H. S. and Blewitt, M. (2013). Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts. Bio-protocol 3(15): e844. DOI: 10.21769/BioProtoc.844.
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分类
癌症生物学 > 通用技术 > 细胞生物学试验
细胞生物学 > 细胞分离和培养 > 转化
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