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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   

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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

  1. Source of tissue: body of embryonic day 13.5 mouse embryos, harvested fresh from pregnant females
  2. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 )
  3. Fetal Calf Serum (FCS) (Life Technologies, Gibco®, catalog number: 10437-028 )
  4. Trypsin (Life Technologies, Gibco®, catalog number: 25200056 )
  5. Dulbecco’s Phosphate Buffered Saline (PBS), without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14190-144 )
  6. Retroviral supernatant containing LMP-p53.1224 shRNA construct (Dickins et al., 2005)
  7. Retroviral supernatant containing pWZL-HRasV12 cDNA construct (Serrano et al., 1997)
  8. Hygromycin B (Life Technologies, catalog number: 10687-010 )
  9. Puromycin (Sigma-Aldrich, catalog number: P9620-10ML )
  10. Hexadimethrine bromide/Polybrene (Sigma-Aldrich, catalog number: H9268 )
  11. Polybrene (1,000x stock) (see Recipes)

Equipment

  1. Tissue culture flasks T75 (Greiner Bio-One, catalog number: 658175 )
  2. 10-cm tissue culture dishes (BD Biosciences, Falcon®, catalog number: 353003 )
  3. 21-gauge needles
  4. 5 ml syringes
  5. 37 °C 10% CO2 cell culture incubator
  6. Table-top centrifuge

Procedure

  1. Retroviral supernatants are prepared as previously described, at a titer of 106 to 107 viral particle per ml of viral supernatant (Pear et al., 1993).
    Note: Do not freeze/thaw supernatant, and use within 6 months.
  2. Primary MEFs are generated from embryonic day 13.5 (E13.5) embryos by passing the embryonic body (excluding head, liver and intestines) through a 21-gauge needle and syringe followed by repeated pipetting into a 10-cm tissue culture dish (1 embryo per dish) in 1 ml of DME medium containing 10% (v/v) FCS (DMEM/FCS). It is not necessary to obtain a single cell suspension at this stage, as trypsinisation at later stages will produce a single cell suspension and excessive manipulation at this stage promotes cell death. Add 9 ml of DMEM/FCS and mix to combine.
  3. Primary MEFs are then incubated in 10% CO2 incubator at 37 °C for 2-3 days undisturbed.
  4. MEFs are washed once in PBS, trypsinised, trypsin inhibited with DMEM/FCS and pelleted at 485 g for 5 minutes.
  5. MEFs are split ~1:2 into a T75 tissue culture flask and incubated in 10% CO2 incubator at 37 °C overnight so that cells are ~60-70% confluent the following day.
  6. On the next morning, aspirate the supernatant and wash once with PBS. Combine the retroviral supernatant containing LMP-p53.1224 shRNA, DMEM/FCS and polybrene using the following recipe:
    Retroviral supernatant          1.5 ml (i.e., ~1:7 dilution)
    DMEM/FCS                          8.5 ml
    Polybrene (1,000x stock)     10 μl (4 μg/ml)
    Total                                     10 ml
  7. After ~7-8 h of infection, repeat step 6, and leave the fresh retroviral supernatant overnight.
  8. On the next day, aspirate the supernatant, wash cells once with PBS, replace with fresh DMEM/FCS, and incubate at 37 °C overnight.
  9. On the following day, replace medium with fresh DMEM/FCS containing 5 μg/ml puromycin (LMP-p53.1224 shRNA construct has a puromycin selectable marker), and leave for 2 days, if not confluent. Otherwise, split as necessary.
  10. At the end of puromycin selection on day 3, cells are washed once with PBS, trypsinised and seeded so that cells are ~60-70% confluent in a T75 flask the following day. Culture cells in DMEM/FCS without puromycin and incubate overnight at 37 °C.
  11. On the next day, repeat steps 6-8, but with retroviral supernatant containing pWZL-HRasV12 cDNA. The two tranductions should be performed sequentially, as suggested, so that p53 knockdown and immortalization precedes HRasV12 overexpression. This ensures the best efficiency of transformation since HRasV12 overexpression with inefficient p53 knockdown results in senescence.
  12. On the following day, replace medium with fresh DMEM/FCS containing 300 μg/ml hygromycin (pWZL-HRasV12 cDNA construct has a hygromycin selectable marker) for 6 days. Replace with fresh hygromycin after 3 days, and split cells when necessary.
  13. At the end of hygromycin selection on day 7, replace with fresh DMEM/FCS without hygromycin.
  14. Passage cells as necessary for another 10-14 days to allow HRasV12 to drive cell proliferation. These transformed cells can now be used for in vitro or in vivo experiments. For example, cells can be injected subcutaneously into the flank of nude mice to assess tumour growth rate in vivo. The cells can be frozen and stored in liquid nitrogen, or can be continuously passaged, however extended passaging will result in additional genetic aberrations based on the knockdown of p53.

Recipes

  1. 1,000x stock polybrene (4 mg/ml)
    Mix 0.2 g of hexadimethrine bromide with 50 ml Milli Q H2O  
    Filter sterilize (0.22 μm)
    Aliquot and store at -20 °C.

Acknowledgments

This protocol was previously used and adapted from Leong et al. (2013).

References

  1. Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J. and Lowe, S. W. (2005). Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37(11): 1289-1295.
  2. Leong, H. S., Chen, K., Hu, Y., Lee, S., Corbin, J., Pakusch, M., Murphy, J. M., Majewski, I. J., Smyth, G. K., Alexander, W. S., Hilton, D. J. and Blewitt, M. E. (2013). Epigenetic regulator Smchd1 functions as a tumor suppressor. Cancer Res 73(5): 1591-1599.
  3. Pear, W. S., Nolan, G. P., Scott, M. L. and Baltimore, D. (1993). Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90(18): 8392-8396. 
  4. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. and Lowe, S. W. (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88(5): 593-602.

简介

细胞转化是人工诱导细胞在体内形成肿瘤的广泛使用的方法。 在这里,我们描述了用于移植到免疫缺陷型裸鼠中的小鼠胚胎成纤维细胞(MEF)的恶性转化的方法,如Leong等人所使用的。 (2013年)。 两步过程包括:1)使用短发夹RNA(shRNA)下调Trp53表达; 和2)致癌HRas V12蛋白的过表达。 Trp53表达的减少导致细胞永生化,并且随后的致癌HRas V12的过表达导致细胞的恶性转化。

关键字:转型, 小鼠胚胎成纤维细胞, hrasv12, p53基因敲除

材料和试剂

  1. 组织来源:胚胎日13.5小鼠胚胎的胚胎,从怀孕雌性新鲜收获的
  2. Dulbecco's Modified Eagle's Medium(DMEM)(Life Technologies,Gibco ,目录号:41965-039)
  3. 胎牛血清(FCS)(Life Technologies,Gibco ,目录号:10437-028)
  4. 胰蛋白酶(Life Technologies,Gibco ,目录号:25200056)
  5. 不含Ca 2+和Mg 2+的Dulbecco's磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:14190-144 )
  6. 含有LMP-p53.1224 shRNA构建体的逆转录病毒上清液(Dickins等人,2005)
  7. 含有pWZL-HRas V12 cDNA构建体(Serrano等人,1997)的逆转录病毒上清液
  8. 潮霉素B(Life Technologies,目录号:10687-010)
  9. 嘌呤霉素(Sigma-Aldrich,目录号:P9620-10ML)
  10. 己二胺溴化物/聚凝胺(Sigma-Aldrich,目录号:H9268)
  11. Polybrene(1,000x原料)(参见配方)

设备

  1. 组织培养瓶T75(Greiner Bio-One,目录号:658175)
  2. 10-cm组织培养皿(BD Biosciences,Falcon ,目录号:353003)
  3. 21号针
  4. 5ml注射器
  5. 37℃,10%CO 2细胞培养箱中
  6. 台式离心机

程序

  1. 如前所述制备逆转录病毒上清液,滴度为10 6至10 7 sup/>病毒颗粒/ml病毒上清液(Pear et al。,1993)。
    注意:不要冷冻/融化上清液,并在6个月内使用。
  2. 通过使胚胎体(不包括头,肝和肠)通过21号针和注射器,然后重复吸入10cm组织培养皿(1个胚胎)中,从胚胎第13.5天(E13.5)胚胎产生初级MEF每皿)在1ml含有10%(v/v)FCS(DMEM/FCS)的DME培养基中。在该阶段不需要获得单一细胞悬浮液,因为在较晚阶段的胰蛋白酶化将产生单细胞悬浮液,并且在该阶段的过度操作促进细胞死亡。加入9ml DMEM/FCS并混合混合。
  3. 然后将原代MEF在10%CO 2培养箱中在37℃温育2-3天,不受干扰。
  4. MEF在PBS中洗涤一次,胰蛋白酶化,用DMEM/FCS抑制胰蛋白酶,并在485g下沉淀5分钟。
  5. 将MEF分裂〜1:2到T75组织培养烧瓶中并在37℃下在10%CO 2培养箱中温育过夜,使得第二天细胞约60-70%汇合。
  6. 第二天早上,吸出上清液并用PBS洗涤一次。使用以下配方组合含有LMP-p53.1224 shRNA,DMEM/FCS和聚凝胺的逆转录病毒上清液:
    逆转录病毒上清液          1.5ml(,〜1:7稀释) DMEM/FCS                          8.5 ml
    Polybrene(1,000x股票)     10μl(4μg/ml)
    总计                                   10 ml
  7. 在〜7-8小时的感染后,重复步骤6,并将新鲜的逆转录病毒上清液过夜。
  8. 第二天,吸出上清液,用PBS洗涤细胞一次,用新鲜的DMEM/FCS替换,并在37℃孵育过夜。
  9. 第二天,用含有5μg/ml嘌呤霉素的新鲜DMEM/FCS(LMP-p53.1224 shRNA构建体具有嘌呤霉素选择标记)替换培养基,如果不汇合,放置2天。否则,根据需要拆分。
  10. 在第3天嘌呤霉素选择结束时,用PBS洗涤细胞一次,胰蛋白酶化并接种,使得细胞在第二天在T75烧瓶中约60-70%汇合。在不含嘌呤霉素的DMEM/FCS中培养细胞,并在37℃下孵育过夜。
  11. 在第二天,重复步骤6-8,但使用含有pWZL-HRas V12 cDNA的逆转录病毒上清液。如所建议的,这两个转录应该顺序进行,使得p53敲低和永生化先于HRas V12 过表达。这确保了转化的最佳效率,因为具有低效p53敲低的HRas V12 过表达导致衰老。
  12. 在第二天,用含有300μg/ml潮霉素的新鲜DMEM/FCS(pWZL-HRas V12 cDNA构建体具有潮霉素选择性标记)替换培养基6天。 3天后更换为新鲜潮霉素,必要时分裂细胞。
  13. 在第7天潮霉素选择结束时,更换为不含潮霉素的新鲜DMEM/FCS。
  14. 根据需要通过细胞另外10-14天以允许HRas V12 驱动细胞增殖。 这些变换的细胞现在可以用于 > r 或体内实验。 例如,可以将细胞皮下注射到裸鼠的侧腹以评估体内肿瘤生长速率。 细胞可以冷冻并储存在液氮中,或者可以连续传代,然而延长的传代将导致基于p53敲低的额外的遗传畸变。

食谱

  1. 1,000x聚苯乙烯(4mg/ml) 将0.2g溴化己二胺与50ml Milli Q H 2 O混合 过滤灭菌(0.22μm)
    等分并储存在-20°C

致谢

此协议之前使用和改编自Leong等人(2013)。

参考

  1. Dickins,R.A.,Hemann,M.T.,Zilfou,J.T.,Simpson,D.R.,Ibarra,I.,Hannon,G.J。和Lowe,S.W。(2005)。 使用稳定和调节的合成microRNA前体检测肿瘤表型。 Nat Genet 37(11):1289-1295。
  2. Leong,HS,Chen,K.,Hu,Y.,Lee,S.,Corbin,J.,Pakusch,M.,Murphy,JM,Majewski,IJ,Smyth,GK,Alexander,WS,Hilton,DJ和Blewitt ,ME(2013)。 表观遗传调节因子Smchd1 作为肿瘤抑制因子。 73(5):1591-1599。
  3. Pear,W.S.,Nolan,G.P.,Scott,M.L。和Baltimore,D。(1993)。 通过瞬时转染产生高滴度无辅助病毒的逆转录病毒。 Proc Natl Acad Sci USA 90(18):8392-8396。 
  4. Serrano,M.,Lin,A.W.,McCurrach,M.E.,Beach,D.and Lowe,S.W。(1997)。 致癌性 ras 会引起与p53和p16的积累有关的过早细胞衰老。 sup> INK4a 。 Cell 88(5):593-602。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
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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Steven Shen
Duke University
Dear Drs. Huei San Leong and Marnie Blewitt,
Hello!
After transduction the prepared Retrovirus Mediated Malignant Transformation of MEF to mouse, in wild type created neoplasm and dramatic tumor grow, however, it could not create in a gene Knockout mouse. What is the possibility, or reason and how to resolve the problem?
If shRNA-p53 transfection is a must step in the oncogenic c-myc puro seleated Myc MEFs or oncogenic Ras puro selected Ras MEFs?

Thanks for your comment and help!
9/13/2014 8:56:43 AM Reply
Marnie Blewitt
Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Australia

Dear Steven,

The p53 knockdown is essential prior to oncogenic Ras transduction, as otherwise Ras with cause senescence.

With regards to why your knockout cells don't create a neoplasm, there are a number of possibilities. Were the knockout cells passaged, transduced and selected alongside the controls? In this way, can you be sure they were properly transformed? If so, it is possible that the knockout cells cannot grow in vivo. Potentially this could be because the gene that has been knocked out is downstream of Ras, meaning even with Ras overexpression you don't transform the cells.

9/14/2014 3:54:26 PM


shobhit mishra
TMU
WHAT WILL BE THE EXPECTED ESTIMATE TO DO THIS EXPERIMENT IN INDIA
6/10/2014 2:48:23 AM Reply
Bio-protocol Editorial Team
bio-protocol.org

Hi shobhit,

We would suggest that your question could be more specific so that it would be answered more efficiently.

Thanks,
Bio-protocol Editorial Team

6/10/2014 1:58:03 PM