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[Bio101] Transfection of S2 Cell with DNA Using CellFectin Reagent
[Bio101] 用脂质体转染试剂转染DNA到S2细胞中   

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Abstract

This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition of copper sulfate, which can then be purified and used as desired. This protocol provides an example to finding out when your peak protein production occurs and a method for determining optimal selection conditions.

Materials and Reagents

  1. Drosophila Schneider 2 (S2) cells (Life Technologies, Invitrogen™, catalog number: R690-07 )
  2. Dros-SFM (Life Technologies, Invitrogen™, catalog number: 10797-025 )
  3. pHASβAG (pRmHA-3 vector with SDF-1β-FLAG inserted. Contains a metallothionein promoter inducible with Cu2+)
  4. pUC-Hsneo (contains the neomycin-resistant cassette required for stable selection)
  5. CellFectin reagent (Life Technologies, Invitrogen™, catalog number: 10362-010 )
  6. FBS (Life Technologies, Invitrogen™, catalog number: 10437-028 )
  7. Penicillin-Streptomycin (P/S) (Life Technologies, Invitrogen™, catalog number: 15140-122 )
  8. G418 sulphate (Life Technologies, Invitrogen™, catalog number: 11811-031 )
  9. CuSO4 (Sigma Aldrich, catalog number: C8027 )
  10. HT supplement (Life Technologies, Invitrogen™, catalog number: 11067-030 )
  11. Transfection solutions (see Recipes)

Equipment

  1. 6-well plate (Corning Incorporated, catalog number: 353046 )
  2. 24-well plate (Corning Incorporated, catalog number: 353047 )

Procedure

  1. Drosophila Schneider 2 (S2) cells are plated out at 1.6 x 106 cells/well (6-well plate, 2 ml /well) and incubated overnight at room temperature (RT).
    Note: The optimal temperature of S2 cells is actually 28 °C and they do not need CO2 to culture.
  2. Following day, cells are washed 2x with Dros-SFM (no FBS, P/S or HT) 1 ml/well.
  3. Prepare the ‘DNA mix’ and ‘CellFectin Mix’ transfection solutions.


  4. 100 μl /sample (i.e. 400 μl total) of the DNA mix is added to 100 μl /sample (i.e. 400 μl total) CellFectin mix. Solution is gently mixed and left at RT for 30 min.
  5. After 30 min, 800 μl /sample (3.2 ml total) of Dros-SFM (no FBS, P/S or HT) is added to the DNA/CellFectin mix.
  6. 1 ml of this solution is then added to the appropriate wells containing the S2 cells and incubated at RT for 5 h.
  7. Following the 5 h incubation, medium is aspirated off and Dros-SFM + 10% FBS + P/S + HT (= complete media) is added to each well (2 ml/well).
  8. Plates are incubated at RT for 48 h.
  9. At 48 h post-transfection the media is removed and replaced with 2 ml media containing G418 sulphate at indicated concentrations. The cells are transferred to 24-well plates (one 6-well ≥ eight 24-wells) supplemented with additional media + G418 to 1 ml.

  10. Cells are incubated at RT with media changed every 5 days (1 ml/well).
  11. After 3 weeks, untransfected cells should be killed by the G418 at least at the highest concentration. The transfected cells should be fine and healthy.
  12. Cells within the 4 μl and 8 μl CellFectin groups are pooled and split 1:1 into two 24-well plates (i.e. 8 wells /plate). After 1 h, media is removed and replaced with either complete SFM + 1.5 mg/ml G418 or complete SFM + 1.5 mg/ml G418 + 1 mM CuSO4.

  13. Cell lysates are taken at 2, 3, 4 and 5 days post-CuSO4 addition to determine peak time of SDF-1β-FLAG production.

Recipes

  1. Transfection solutions
    a.  DNA mix (per sample):                        Total (9 samples)
         4 μg pHASβAG = 1.67 μl                   15.0 μl
         0.2 μg pUC-Hsneo = 0.63 μl                5.7 μl
         100 μl Dros-SFM (no FBS or P/S)         900 μl
    b.  CellFectin Mix (per sample):                Total (4 samples)
         4 μl or 8 μl CellFectin reagent             16 or 32 μl
         100 μl Dros-SFM                               400 μl

References

  1. Bunch, T. A., Grinblat, Y. and Goldstein, L. S. (1988). Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res 16(3): 1043-1061.

简介

该方法提供了用具有选择插入片段(在这种情况下是SDF-1β-FLAG)的pRmHA-3(或相似)载体转染果蝇S2细胞并产生稳定细胞系的逐步指导。 然后,该细胞系能够在诱导条件下通过加入硫酸铜产生目的蛋白,然后可以根据需要纯化和使用。 这个协议提供了一个例子,找出什么时候你的蛋白质峰的产生和一个确定最佳选择条件的方法。

材料和试剂

  1. (S2)细胞(Life Technologies,Invitrogen TM,目录号:R690-07)
  2. Dros-SFM(Life Technologies,Invitrogen TM,目录号:10797-025)
  3. pHASβAG(插入有SDF-1β-FLAG的pRmHA-3载体,含有可用Cu 2+诱导的金属硫蛋白启动子)
  4. pUC-Hsneo(含有稳定选择所需的新霉素抗性盒)
  5. CellFectin试剂(Life Technologies,Invitrogen TM,目录号:10362-010)
  6. FBS(Life Technologies,Invitrogen TM,目录号:10437-028)
  7. 青霉素 - 链霉素(P/S)(Life Technologies,Invitrogen TM,目录号:15140-122)
  8. G418硫酸盐(Life Technologies,Invitrogen TM,目录号:11811-031)
  9. CuSO 4(Sigma Aldrich,目录号:C8027)
  10. HT补充剂(Life Technologies,Invitrogen TM,目录号:11067-030)
  11. 转染解决方案(参见配方)

设备

  1. 6孔板(Corning Incorporated,目录号:353046)
  2. 24孔板(Corning Incorporated,目录号:353047)

程序

  1. 将果蝇Schneider 2(S2)细胞以1.6×10 6个细胞/孔(6孔板,2ml /孔)接种,并在室温(RT)下孵育过夜。 注意:S2细胞的最佳温度实际上是28℃,并且它们不需要CO 2培养。
  2. 第二天,将细胞用Dros-SFM(无FBS,P/S或HT)以1ml /孔洗涤2次。
  3. 准备"DNA混合物"和"CellFectin Mix"转染溶液

  4. 将100μl/样品(即总共400μl)的DNA混合物加入到100μl/样品( i )CellFectin混合物。 将溶液温和混合并在室温下放置30分钟。
  5. 30分钟后,向DNA/CellFectin混合物中加入800μl/样品(总共3.2ml)的Dros-SFM(无FBS,P/S或HT)。
  6. 然后将1ml该溶液加入到含有S2细胞的合适孔中,并在室温下温育5小时。
  7. 孵育5小时后,吸出培养基,向每个孔中加入Dros-SFM + 10%FBS + P/S + HT(=完全培养基)(2ml /孔)。
  8. 将平板在室温下温育48小时。
  9. 转染后48小时,除去培养基并用含有指定浓度的G418硫酸盐的2ml培养基替换。将细胞转移到补充有额外培养基+ G418至1ml的24孔板(一个6孔≥8个24孔)中。

  10. 将细胞在室温下温育,每5天更换一次培养基(1ml /孔)。
  11. 3周后,未转染的细胞应至少以最高浓度被G418杀死。转染的细胞应该是精细和健康的。
  12. 合并4μl和8μlCellFectin组中的细胞,并以1:1混合在两个24孔板中(即8孔/板)。 1小时后,除去培养基并用完全SFM + 1.5mg/ml G418或替换 完全SFM + 1.5mg/ml G418 + 1mM CuSO 4
  13. 在CuSO 4加入后2,3,4和5天取出细胞裂解物,以确定SDF-1β-FLAG产生的峰值时间。

食谱

  1. 转染解决方案
    a。  DNA混合物(每个样品):                    ; 总计(9个样本)
         4μgpHASβAG=1.67μl                  15.0微升
         0.2μgpUC-Hsneo =0.63μl                5.7μl
         100μlDros-SFM(无FBS或P/S)         900μl
    b。  CellFectin Mix(每个样品):              总计(4个样本)
         4μl或8μlCellFectin试剂              16或32微升
         100μlDros-SFM                               400μl

参考文献

  1. Bunch,T.A.,Grinblat,Y.and Goldstein,L.S.(1988)。 在培养的果蝇细胞中表征和使用果蝇金属硫蛋白启动子。 Nucleic Acids Res 16(3):1043-1061。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cronshaw, D. G. (2012). Transfection of S2 Cell with DNA Using CellFectin Reagent. Bio-protocol Bio101: e190. DOI: 10.21769/BioProtoc.190;
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