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[Bio101] A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts
[Bio101] 拟南芥叶肉细胞原生质体瞬时表达体系   

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Abstract

This method can be used to free and separate the mesophyll cells from Arabidopsis leaves. The protoplasts that are generated in this way can be used for transient expression for protein activity and subcellular localization assays.

Keywords: Protoplast(原生质体), Transient expression(瞬时表达), Arabidopsis(拟南芥), Transfection(转染)

 

Materials and Reagents

  1. Arabidopsis (Ecotype: Columbia)
  2. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
  3. PEG4000 (Fluka, catalog number: 81240 )
  4. Mannitol
  5. NaCl
  6. KCl
  7. CaCl2
  8. MgCl2
  9. 0.5 M MES (pH 5.7)
  10. β-mercaptoethanol
  11. Cellulase R10 purchased from http://www.yakult.co.jp/ypi/english/frame03.html
  12. Macerozyme R10 purchased from http://www.yakult.co.jp/ypi/english/frame03.html
  13. Bright-Line/Dark-Line Counting Chambers (catalog numbers: 3100 , 3110, 3200 , 3500 , 1490 , 1492 , 1475 and 1483 )
  14. Enzyme solution (see Recipes)
  15. PEG solution (see Recipes)
  16. W5 solution (see Recipes)
  17. MMg solution (see Recipes)
  18. Washing and incubation solution (see Recipes)

Equipment

  1. IEC clinical centrifuge
  2. Chamber Counter (instruction attached)
  3. Petri plate
  4. Vacuum desiccator
  5. Zeiss LSM510
  6. Aluminum foil
  7. Nylon filters (35-75 µm) (Carolina Biological Supplies, catalog number: 65-2222N )

Procedure

  1. Protoplast Isolation
    Arabidopsis Columbia plants are planted on soil, cold treated for 3 d, and then transferred to the growth chamber (10 h Light/14 h Dark, 22 °C day/ 20 °C night, 80-100 μE). Well expanded leaves from 3.5-4.5 weeks old plants (6-8 leaves with elongated petiole) are used to prepare protoplasts.
    Protoplast isolation procedure:
    a.  Make 10 ml enzyme solution. This is enough for more than 10 standard transfections.  Pour solution into 15 cm petri plate.
    b.  Cut 0.5-1 mm leaf strips with fresh razor blades without wounding. Use 2-4 young leaves per plant. Put strips in enzyme solution immediately after cut. 10 ml enzyme solution can hold 40-60 such leaves.
    c. Put the plate into to a vacuum desiccator and apply vacuum for 30 min. Continue the digestion for about 3 h without shaking in the dark (wrapped in Al foil) at room temperature (RT) 22-25 °C.
    d.  Use a round-bottom tube (like the one used for E. coli culture), filter the enzyme solution containing protoplasts with a 35-75 µm nylon mesh by slowly releasing the cell-containing solution from a 10 ml transfer pipette. Rinse the plate once with 4 ml W5 solution. Combine the filter-through and spin at 100 x g to pellet the protoplasts 1.5 min (speed 3 with an IEC clinical centrifuge).
    e.  Resuspend protoplasts once in 10 ml W5 solution. Spin at speed 3 for 1.5 min, and resuspend in 2 ml W5 solution. Count the cells using a chamber counter. Add more W5 to a cell density of 2.5 x 105/ml.
    f.  Keep the protoplasts on ice (30 min) in W5 solution.
    g. Spin down protoplasts (speed 3 for 1 min) and resuspend in MMg solution (2.5 x 105 /ml) before PEG transfection.
  2. PEG Transfection
    All steps are carried out at RT (e.g. 23 ˚C)
    a.  Coat the 15 ml conical bottom tube with 5% FBS for 1 sec. Spin for 1 min and remove the leftover.
    b.  Add 60 μl DNA (60-120 μg of plasmid DNA of 5 kb in size. For co-transfection, use each with equal moles, the total remains the same).
    c.  Add 400 μl protoplasts to a microfuge tube (1 x 105 protoplasts), mix well gently with a 2 ml plastic transfer pipet.
    d.  Add 460 μl of PEG/Ca solution, mix well (handle 6-10 samples each time) gently with a 2 ml plastic transfer pipet. Incubate at 23 °C for 5-30 min.
    e.  Dilute with 3 ml W5 solution and mix well gently with a 2 ml plastic transfer pipet.
    f.  Spin at speed 3 in a clinical centrifuge for 1 min, remove supernatant. Resuspend protoplasts gently in 200 μl WI solution by a 2 ml plastic transfer pipet.
    g.  Wrap the tubes with Al foil and keep at ~23 °C until microscopic observation.
    h.  Check the cells for fluorescence under microscope. Most cells should be round with flashy red chloroplasts (auto fluorescence) dispersed evenly throughout the cell.
    Common filter settings (on Zeiss LSM510):

     Excitation (nm)
    Emission (nm)
    GFP
    488
    BP505-530
    RFP
    543
    BP560-615
    Chlorophyll
    488
    LP650

Recipes

  1. Enzyme solution (10 ml)
    stock
    volume
    final conc.
    1 M mannitol
    4 ml
    0.4 M
    1 M KCl
    0.2 ml
    20 mM
    0.5 M MES (pH 5.7)
    0.4 ml
    20 mM
    cellulase R10
    100-150 mg
    1-1.5%
    macerozyme R10
    20-40 mg
    0.2-0.4%

    Heat the enzyme solution at 55 °C for 10 min (to inactivate proteases and enhance enzyme solubility) and cool it to RT before adding.
    1 M CaCl2
    0.1 ml
    10 mM
    β-mercaptoethanol
    4 μl
    5 mM
    10% FBS
    0.1 ml
    0.1%

  2. PEG solution (40%, w/v) 10 ml
    PEG4000
    4 g
    40% w/v
    **Very Important!!


    1 M mannitol
    2 ml
    200 mM
    1 M CaCl2
    1 ml
    100 mM
    H2O
    3.5 ml


  3. W5 solution (50 ml)
    1 M NaCl
    7.7 ml
    154 mM
    1 M CaCl2
    6.25 ml
    125 mM
    1 M KCl
    0.25 ml
    5 mM
    0.5 M MES-K (pH 5.7)
    0.2 ml
    2 mM

  4. MMg solution (5 ml)
    1 M mannitol
    2 ml
    0.4 M
    0.3 M MgCl2
    0.25 ml
    15 mM
    0.5 M MES-K (pH 5.7)
    40 μl
    4 mM

  5. Washing and incubation solution (WI) 10 ml
    final conc.
    stock
    volume
    1 M mannitol
    5 ml
    0.5 M
    0.5 M MES (pH 5.7)
    80 μl
    4 mM
    1 M KCl
    0.2 ml
    20 mM

  6. Directions for Chamber Counter
    http://www.hausserscientific.com/
    Bright-Line / Dark-Line Counting Chambers
    Catalog Numbers: 3100, 3110, 3200, 3500, 1490, 1492, 1475 and 1483
    Usage: Cell Counts
    Cell Depth: 0.100mm +/- 2% (1/10 mm)

    Volume: 0.1 Microliter
    Ruling Pattern: Improved Neubauer, 1/400 Square mm
    Rulings cover 9 square millimeters. Boundary lines of the Neubauer ruling are the center lines of the groups of three (these are indicated in the illustration below). The central square millimeter is ruled into 25 groups of 16 small squares, each group separated by triple lines, the middle one of which is the boundary. The ruled surface is 0.10 mm below the cover glass, so that the volume over each of the 16 small squares is.00025 cubic mm.
    The number of cells per milliliter = Number of cells counted per square millimeter X dilution (if used) X 10,000


    Neubauer Ruling

Acknowledgments


This protocol is consolidated from Jen Sheen’s protocol and Inhwan Hwang’s protocol. For references please go to the following websites for their publication lists:
http://genetics.mgh.harvard.edu/sheenweb/
http://www.postech.ac.kr/center/cpit/professor.html

References

  1. Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F. and Sze, H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiol 147(4): 1675-1689.

简介

该方法可用于从拟南芥叶中游离和分离叶肉细胞。 以这种方式产生的原生质体可用于蛋白质活性和亚细胞定位测定的瞬时表达。

关键字:原生质体, 瞬时表达, 拟南芥, 转染

 

材料和试剂

  1. 拟南芥(生态型:哥伦比亚)
  2. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F6178)
  3. PEG4000(Fluka,目录号:81240)
  4. 甘露醇
  5. NaCl
  6. KCl
  7. CaCl 2
  8. MgCl 2
  9. 0.5 M MES(pH 5.7)
  10. β-巯基乙醇
  11. Cellulase R10购自 http://www.yakult.co.jp/ypi/english /frame03.html
  12. Macerozyme R10购自 http://www.yakult.co.jp/ypi/english /frame03.html
  13. 亮线/暗线计数室(目录号:3100,3110,3200,3500,1490,1492,1475和1483)
  14. 酶溶液(见配方)
  15. PEG溶液(见配方)
  16. W5解决方案(参见配方)
  17. MMg解决方案(参见配方)
  18. 洗涤和孵育溶液(见配方)

设备

  1. IEC临床离心机
  2. 室内计数器
  3. 培养皿
  4. 真空干燥器
  5. Zeiss LSM510
  6. 铝箔
  7. 尼龙过滤器(35-75μm)(Carolina Biological Supplies,目录号:65-2222N)

程序

  1. 原生质体隔离
    将拟南芥哥伦比亚植物种植在土壤上,冷处理3天,然后转移到生长室(10小时光照/14小时黑暗,22℃白天/20℃夜晚,80-100℃) μE)。 来自3.5-4.5周龄植物的膨大叶 (6-8叶与细长的叶柄)用于准备原生质体 原生质体分离程序:
    a。 制成10ml酶溶液。这足以进行10次以上的标准转印。将溶液倒入15cm培养皿中。
    b。 用新鲜剃刀刀片切割0.5-1毫米的叶条,无伤口。每株使用2-4片叶。在切割后立即将条带放入酶溶液中。 10ml酶溶液可以容纳40-60个这样的叶子 C。将板放入真空干燥器中并施加真空30分钟。在室温(RT)22-25℃下,在黑暗中不振摇(包裹在Al箔中)继续消化约3小时。
    d。使用圆底管(如用于大肠杆菌培养物的管),通过从10ml移液管缓慢释放含细胞的溶液,用35-75μm尼龙网过滤含有原生质体的酶溶液。用4 ml W5溶液冲洗板一次。将过滤器和旋转以100×g混合以使原生质体沉淀1.5分钟(使用IEC临床离心机的速度3)。
    e。 重悬原生质体在10ml W5溶液中一次。以速度3旋转1.5分钟,并重悬于2ml W5溶液中。使用室计数器计数细胞。将更多的W5添加到2.5×10 5个/ml/ml的细胞密度 f。 保持原生质体在冰上(30分钟)在W5溶液 G。旋转原生质体(速度3 1分钟),并在PEG转染前重悬于MMg溶液(2.5×10 5/ml)中。
  2. PEG转染
    所有步骤都在RT(例如 .23˚C)下进行。
    a。 用15%锥形底部管用5%FBS涂覆1秒。旋转1分钟,清除残留物。
    b。 加入60微升DNA(60-120微克5 kb大小的质粒DNA,对于共转染,使用等摩尔的,总数保持不变)。
    c。 添加400微升原生质体到微量离心管(1×10 5个原生质体),用2毫升塑料移液管轻轻混合。
    d。加入460微升的PEG/Ca溶液,混匀(每次处理6-10样品)轻轻用2毫升塑料移液管。在23℃孵育5-30分钟。
    e。 用3ml W5溶液稀释,并用2ml塑料移液管轻轻混匀 f。  在临床离心机中以速度3旋转1分钟,除去上清液。 通过2 ml塑料移液管在200μlWI溶液中轻轻重悬原生质体 g。 用铝箔包裹管,保持在〜23°C直到显微镜观察 h。  检查细胞的显微镜下的荧光。 大多数细胞应该是闪烁的红色叶绿体(自动荧光)均匀分散在整个细胞周围 常用过滤器设置(在Zeiss LSM510上):

     激发(nm)
    发射(nm)
    GFP
    488
    BP505-530
    RFP
    543
    BP560-615
    叶绿素
    488
    LP650

食谱

  1. 酶溶液(10ml)
    股票
    音量
    最终浓度。
    1 M甘露醇
    4 ml
    0.4 M
    1 M KCl
    0.2 ml
    20 mM
    0.5 M MES(pH 5.7)
    0.4 ml
    20 mM
    纤维素酶R10
    100-150 mg
    1-1.5%
    macerozyme R10
    20-40 mg
    0.2-0.4%

    将酶溶液在55℃加热10分钟(以灭活蛋白酶并增强酶溶解度),并在加入前将其冷却至室温。
    1 M CaCl 2
    0.1 ml
    10 mM
    β-巯基乙醇 4微升
    5 mM
    10%FBS
    0.1 ml
    0.1%

  2. PEG溶液(40%,w/v)10ml
    PEG4000
    4克
    40%w/v
    ** 非常重要!


    1 M甘露醇
    1 M NaCl
    7.7 ml
    154 mM
    1 M CaCl 2
    6.25 ml
    125 mM
    1 M KCl
    0.25 ml
    5 mM
    0.5 M MES-K(pH 5.7)
    0.2 ml
    2 mM

  3. MMg溶液(5ml)
    1 M甘露醇
    2 ml
    0.4 M
    0.3 M MgCl 2/
    0.25 ml
    15 mM
    0.5 M MES-K(pH 5.7)
    40微升
    4 mM

  4. 洗涤和孵育溶液(WI)10ml
    最终浓度。
    股票
    音量
    1 M甘露醇
    5 ml
    0.5 M
    0.5 M MES(pH 5.7)
    80μl
    4 mM
    1 M KCl
    0.2 ml
    20 mM

  5. 会议室
    的指示 http://www.hausserscientific.com/
    明线/暗线计数室
    目录号:3100,3110,3200,3500,1490,1492,1475和1483
    用法:单元格计数
    单元深度:0.100mm +/- 2%(1/10mm)
    体积:0.1微升
    裁定模式:改进的Neubauer,1/400平方毫米
    裁决覆盖9平方毫米。纽鲍尔线的边界线是三个组的中心线(这些在下面的图示中示出)。中心方形毫米被划分为25组16个小正方形,每组由三条线分隔开,中间一条是边界。刻度表面在盖玻片下方0.10mm,使得16个小正方形中的每一个上的体积为0.00025立方毫米。
    每毫升的细胞数=每平方毫米X稀释(如果使用)计数的细胞数×10,000


    Neubauer裁决

致谢


该协议从Jen Sheen的协议和Inhwan Hwang的协议合并。 有关参考信息,请访问以下网站以获取其出版物列表:
http://genetics.mgh.harvard.edu/sheenweb/
http://www.postech.ac.kr/center/cpit/professor.html

参考文献

  1. Li,X.,Chanroj,S.,Wu,Z.,Romanowsky,S.M.,Harper,J.F。和Sze,H。(2008)。 不同的内体Ca 2 + /Mn 2+ 泵通过分泌过程影响根生长。 植物生理学 147(4):1675-1689。
  • English
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. (2011). A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts. Bio-protocol Bio101: e70. DOI: 10.21769/BioProtoc.70;
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