我们热爱生命科学!-生物行
当前位置: 主页 > English > Technology > Methods > Cell Culture

Transient Expression Assay Using Arabidopsis Mesophyll Proto

时间:2006-07-24 14:10来源:Scienceboard.net 作者:admin
Thursday, December 04, 2003

Description
Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts : Protoplast Isolation

Procedure
Plant Materials:
BE plants grown on the B5 medium
Greenhouse-grown BE, Col, Ler and C24 plants are fine
Use well expanded leaves from 3-4 weeks old plants (the second and/or third/fourth pair, 1-2 cm) before flowering. Shorter photoperiod (12-13 h light or less) is recommended for Col and Ler that flower earlier under long-day condition.

Protoplast Isolation Procedure:
Cut 0.5-1 mm leaf strips with fresh razor blades without wounding. This is perhaps the most tedious part for most people. However, I consider it easier and more efficient than peeling the lower epidermis of the leaves one by one. It takes some practice to do a good job in cutting leaves. A very good preparation yields around 107 protoplasts/g fresh weight (about 100 to 150 leaves digested in 40-60 ml of enzyme solution). For a practice or small scale experiments, 10-20 leaves digested in 5-10 ml cellulase/macerozyme solution will give 0.5 -1 x 106 protoplasts that are enough for more than 50-100 samples (1-2 x 104 protoplasts per sample). Note that it is not necessary to use 106 protoplasts per sample for gene expression analysis as commonly recommended in other protoplast protocols. The experiments can be easily scaled up or down as long as the recommended DNA/protoplast ratio is followed (see below).

Use a flask (125-ml flask for 10 ml enzyme solution) with a side arm for leaf digestion and apply vacuum infiltration for 5-30 min or just put leaf strips in a Petri dish with enzyme solution and put it into to a vacuum desiccator. Make sure that the leaf strips are submerged in the enzyme solution. Continue the digestion for another 60 to 90 min with gentle shaking (40 rpm on a platform shaker) or for 3 to 12 h without shaking in the dark (depending on the experimental goals and desirable responses). This step needs to be tested empirically for your own assay. The usual prolonged incubation of leaves for 16-18 h in the dark for protoplast isolation is stressful and may eliminate physiological responses of leaf cells. However, the stress condition may be potentially important for the dedifferentiation and regeneration processes. The enzyme solution should now turn green which indicates the release of rotoplasts (check under microscope. the size of Arabidopsis mesophyll protoplasts is around 30 to 50 mm). Release the protoplasts by shaking at 80 rpm for 1 min. We do not intend to release protoplasts 100%. Be gentle with the protoplasts!

Filter the enzyme solution containing protoplasts with a 35-75 mm nylon mesh. Spin at 100 x g to pellet the protoplasts in a round-bottomed tube for 1-2 min (e.g., speed 3 with an IEC clinical centrifuge). Higher speed or the addition of CaCl2 (50 mM) may be used if the protoplast recovery is poor. The pelleted protoplasts should be resuspended easily by gentle shaking. Wash protoplasts once in cold washing/incubation (WI) solution for electroporation or W5 solution for PEG transfection. Resuspend protoplasts in the same solution at 2 x 105/ml. Keep the protoplasts on ice for 30 min in WI or W5 solution before transfection. For some experiments, protoplasts could be kept at room temperature before use (Please test). Although the protoplasts can be kept on ice for at least 24 h, freshly prepared protoplasts should be used for the study of regulated gene expression, signal transduction, and protein trafficking, processing and localization. We use these protoplasts to study leaf cell responses to sugars, auxin, ABA, cytokinin, heat, EtOH, H2O2, calcium, and elicitors. The responses in protoplasts are similar to those observed in intact plant leaves. These protoplasts are also a good source for the isolation of intact nuclei and chloroplasts. If W5 solution is used, spin down protoplasts and resuspend in MMg solution (2 x 105/ml) before PEG transfection.


Recipes
Enzyme solution
1-1.5 % cellulase R10 (RS is too strong)
0.2-0.4% macerozyme R10 (Yakult Honsha, Tokyo, Japan)
0.4 M mannitol
20 mM KCl
20 mM MES, pH 5.7
Heat the enzyme solution at 55oC for 10 min (to inactivate proteases and enhance enzyme solubility) and cool it to room temperature before adding
10 mM CaCl2
5 mM b-mercaptoethanol (optional)
0.1% BSA (Sigma A-6793)
The enzyme solution is light brown but clear (passed through a 0.45 mm filter).

PEG solution (40%, v/v)
4 g PEG4000 (Fluka, #81240)
3 ml H2O
2.5 ml 0.8 M mannitol
1 ml 1M Ca(NO3)2 or CaCl2

Washing and incubation solution (WI)
0.5 M mannitol,
4 mM MES, pH 5.7
20 mM KCl

W5 solution
154 mM NaCl
125 mM CaCl2
5 mM KCl
2 mM MES (pH 5.7) (no glucose since we use glucose as a signal)

MMg solution
0.4 M mannitol
15 mM MgCl2
4 mM MES (pH 5.7)


Supplies


Tips

(责任编辑:泉水)
顶一下
(0)
0%
踩一下
(0)
0%
------分隔线----------------------------
发表评论
请自觉遵守互联网相关的政策法规,严禁发布色情、暴力、反动的言论。
评价:
表情:
用户名: 验证码:点击我更换图片