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[Bio101] VSVG Psudotyped Retrovirus Production
[Bio101] VSVG假型逆转录病毒生产方案   

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Abstract

Retrovirus vector pseudotyped with vesicular stomatitis virus G (VSV-G) protein has been proven to exhibit high efficiency to deliver genes in a variety of cells. Efficiency is affected by relative cell growth rate and phosphatidylserine level on the cell membrane.

Materials and Reagents

  1. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11995-065 )
  2. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
  3. Penicillin/streptomycin solution (Life Technologies, Gibco®, catalog number: 15140-122 )
  4. NaCl (Sigma-Aldrich, catalog number: S7653 )
  5. HEPES (Sigma-Aldrich, catalog number: H7523 )
  6. Na2HPO4 (Sigma-Aldrich, catalog number: S7907 )
  7. CaCl2 (Sigma-Aldrich, catalog number: C5080 )
  8. Chloroquine (Sigma-Aldrich, catalog number: C6628 )
  9. Sodium butyrate (Sigma-Aldrich, catalog number: B5887 )
  10. Gag/pol (Cell Biolabs, catalog number: RV-111 )
  11. VSVG and retrovirus vector (Clontech, catalog number: 631512 )
  12. Calcium-phosphate (Ca-P) transfection solution (see Recipes)
  13. 2x HeBS (pH 7.0) (see Recipes)
  14. DMEM culture medium (see Recipes)

Equipment

  1. 24-Well plate
  2. Centrifuges
  3. Water bath
  4. 10 cm tissue culture plate
  5. 0.45 micro filter

Procedure

  1. Day 1
    Plate healthy 293 cells in 10 cm tissue culture plate (7 x 106 cell/well in 10 ml culture medium).

  2. Day 2
    1. Prepare for Ca-P (calcium-phosphate) transfection solution.
    2. Add 2x HeBS (pH 7.0) 450 μl to the Ca-P transfection solution and continually mix by vortexing.
    3. Replace cell medium with fresh DMEM culture medium containing 25 μM final concentration of chloroquine.
    4. Sprinkle the 900 μl retrovirus vector mixture from step on cells and incubate cells at 37 °C.
    5. After 3-4 h, replace cell medium with fresh DMEM culture medium, add sodium butyrate at final concentration of 10 μM and incubate at 37 °C.
    Note: Must be removed after 12-14 h.

  3. Day 3
    Replace the DMEM culture medium containing sodium butyrate with fresh DMEM culture medium. Incubate cells at 32 °C O/N.

  4. Day 4
    Harvest viral supernatant and store it at 4 °C. Then add fresh DMEM culture medium to the cells and continue to incubate cells at 32 °C.

  5. Day 5
    Repeat day 4. Place healthy 293 cells for titering next day.

  6. Day 6
    Repeat day 4. Combine all the supernatant together and filtered through 0.45 micron filter. (If high tittering virus is required, spin the supernatant at 25,000 RPM for 3 h at 4 °C. Remove supernatant carefully, and resuspend viral pellet in less than 0.5 ml of supernatant.)

  7. Add 5 μl of concentrated virus to 293 cells (50% confluent) for tittering test. Snap-freeze by dry ice/ethanol concentrated virus and store at -80 °C.

Recipes

  1. DMEM culture medium
    Supplemented with
    10% FBS
    1% Penicillin/streptomycin solution
  2. 2x HeBS (pH 7.0)
    0.28 M final NaCl
    0.05 M final HEPES
    1.5 mM final Na2HPO4
    Adjusted to pH 7.0
  3. Mix the following for Ca-P transfection solution
    54 μl 2 M CaCl2
    15 μg gag/pol (if use package cell line which expresses gag/pol, no need to add it)
    6 μg VSVG
    9 μg retrovirus vector
    Use sterile water to make total volume to 450 μl and mix well.

简介

已经证明用水泡性口炎病毒G(VSV-G)蛋白假型包装的逆转录病毒载体表现出在多种细胞中递送基因的高效率。 效率受相对细胞生长速率和细胞膜上的磷脂酰丝氨酸水平的影响。

材料和试剂

  1. Dulbecco's改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11995-065)
  2. 胎牛血清(FBS)(Gemini Bio-Products,目录号:900-108)
  3. 青霉素/链霉素溶液(Life Technologies,Gibco ,目录号:15140-122)
  4. NaCl(Sigma-Aldrich,目录号:S7653)
  5. HEPES(Sigma-Aldrich,目录号:H7523)
  6. Na 2 HPO 4(Sigma-Aldrich,目录号:S7907)
  7. CaCl 2(Sigma-Aldrich,目录号:C5080)
  8. 氯喹(Sigma-Aldrich,目录号:C6628)
  9. 丁酸钠(Sigma-Aldrich,目录号:B5887)
  10. Gag/pol(Cell Biolabs,目录号:RV-111)
  11. VSVG和逆转录病毒载体(Clontech,目录号:631512)。
  12. 磷酸钙(Ca-P)转染溶液(参见配方)
  13. 2x HeBS(pH 7.0)(参见配方)
  14. DMEM培养基(见配方)

设备

  1. 24孔板
  2. 离心机
  3. 水浴
  4. 10厘米组织培养板
  5. 0.45微过滤器

程序

  1. 第1天
    将在10cm组织培养板(7×10 6个细胞/孔,在10ml培养基中)中的健康293细胞平板。

  2. 第2天
    1.准备Ca-P(磷酸钙)转染溶液 2.向Ca-P转染溶液中加入450μl2x HeBS(pH 7.0),并通过涡旋持续混合。
    3.用含有25μM终浓度氯喹的新鲜DMEM培养基替换细胞培养基 4.将来自步骤的900μl逆转录病毒载体混合物洒在细胞上,并在37℃下孵育细胞。
    5. 3-4小时后,用新鲜的DMEM培养基更换细胞培养基,加入终浓度为10μM的丁酸钠并在37℃下培养。
    注意:必须在12-14小时后移除。

  3. 第3天
    用新鲜的DMEM培养基更换含有丁酸钠的DMEM培养基。在32°C O/N孵育细胞。

  4. 第4天
    收获病毒上清,并存储在4°C。然后向细胞中加入新鲜的DMEM培养基,继续在32℃孵育细胞
  5. 第5天
    重复第4天。将健康293细胞用于第二天的滴度
  6. 第6天
    重复第4天。将所有上清液合并在一起,并通过0.45微米过滤器过滤。 (如果需要高滴度病毒,在4℃下以25,000RPM旋转上清液3小时,小心除去上清液,并在小于0.5ml上清液中重悬病毒沉淀)。

  7. 加入5μl浓缩的病毒到293细胞(50%汇合)进行滴定试验。通过干冰/乙醇浓缩病毒快速冷冻并储存在-80℃

食谱

  1. DMEM培养基
    补充
    10%FBS
    1%青霉素/链霉素溶液
  2. 2x HeBS(pH 7.0)
    0.28M最终NaCl 0.05 M最终HEPES
    1.5mM最终Na 2 HPO 4水溶液 调至pH 7.0
  3. 混合以下用于Ca-P转染溶液
    54μl2 M CaCl 2 15μggag/pol(如果使用表达gag/pol的包装细胞系,则无需添加) 6μgVSVG
    9μg逆转录病毒载体
    使用无菌水使总体积为450μl,混匀
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, J. (2011). VSVG Psudotyped Retrovirus Production. Bio-protocol Bio101: e158. DOI: 10.21769/BioProtoc.158;
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