Transient Expression Assay Using Maize Mesophyll Protoplasts

Thursday, December 04, 2003

Description
Transient Expression Assay Using Maize Mesophyll Protoplasts

Procedure
Plant Growth
Soak seeds in water for overnight. Use half vermiculite and half pelite moss and germinate seeds under light for 3 days (1 cm shoots visible) before moving to the dark chamber. Grow plants in the dark until the second leaf is about 10-15 cm above the 1st leaf.. It takes about 10 to 11 days at 25 oC. Only give water but no nutrients (lots of goodies in the seed) because excess salts can make the manipulation of mesophyll protoplasts more difficult. It is not necessary to grow plants under sterile conditions and perform surface sterilization. Amp (50-100 mg/ml) can be added to the culture medium to prevent bacterial growth for longer incubation (e.g., 16 h). Etiolated or greening leaf protoplasts will be used for most of the experiments. Seedling stems and roots can also be used. Avoid using green (light-grown) leaves which give fragile protoplasts that are difficult to manipulate and have low photosynthetic gene activity except in a narrow window during leaf development.

Protoplast Isolation
Cut the middle part (6-8 cm) of the second leaves. Pile 20 leaves (press gently) and cut 0.5 mm strips without bruising (wounding) the leaves. This is perhaps the most difficult part for most people. It takes practice. I usually prepare several fresh razor blades (rinsed with 95% ethanol) and change razor blades when a dull edge can be sensed. A good preparation yields around 5 x106 protoplasts/g fresh weight which are enough for at least 50 electroporation samples (105 each). I have scaled it up by using fewer protoplasts (103-4 per sample with BTX 96-well electroporator (maximal 5000 samples can be carried out).

Use a flask (250 ml flask for 10-20 ml enzyme solution) with a side arm for leaf digestion and apply vacuum for 30 min for infiltration. Continue the digestion for another 120 min with gentle shaking (40 rpm on a platform shaker). The usual prolonged incubation of leaves for 16-18 h in the dark for protoplast isolation is stressful and might eliminate physiological responses of leaf cells (e.g., light responses). The enzyme solution should now turn green for greening leaves and yellow for etiolated leaves grown in the dark which indicates the release of round protoplasts (check under microscope, the size of maize mesophyll protoplasts is around 25 to 35 mm). Release the protoplasts thoroughly by shaking at 80 rpm for 5 min. No intention is made to release protoplasts 100%.

Filter the enzyme solution containing protoplasts with a 35 mm nylon mesh. Spin at 150 x g to pellet the protoplasts in a round-bottomed tube for 2 min (speed 3-4 with an IEC clinical centrifuge). Higher speed may be used if the protoplast recovery is poor. The protoplast pellet should be resuspended easily by gentle shaking. Wash protoplasts once in cold electroporation solution and resuspend in the same solution at 1-2 x 106/ml. Keep the protoplasts on ice. (However, keep all steps at room temperature if cold treatment will abolish the desired physiological responses; e.g. phytochrome response). Although the protoplasts can be kept on ice for at least 24 h, freshly prepared protoplasts should be used for the study of gene regulation, signal transduction, and protein trafficking, processing, and localization. These protoplasts are also a good source for the isolation of intact nuclei and chloroplasts.

Electroporation
Use 1-2 x 105 protoplasts per electroporation in 300 ml with 25 to 50 mg of plasmid DNA of 5 kb in size (10 mg/kb). The use of carrier DNA is unnecessary. The optimal electroporation condition is 5 msec, 400 V and 200 mF, and one pulse. Two to three pulses can be used if an extremely high transfection efficiency (50-80%) is desired. Promega X-cell 450 (no longer available), Hoefer Progenetor II, BTX T-820, Biorad Gene Pulser with a capacitance extender and even a home-made machine can provide the condition. Keep electroporated samples on ice for 10 min before resuspension in a 6-well tissue culture dish (Falcon 3046) at 0.5-2 x 105/ml. The dish can be coated with 5% calf serum for 10 sec before use to prevent the sticking of the protoplasts to the plastic surface (BSA is not good for this purpose). The protoplast incubation time is 3-6 h for RNA analysis and labeling, and 6-24 h for enzyme activity analysis and protein labeling. About 103-104 protoplasts are enough for enzyme assay (depending on promoter strength) but 0.5 x 106 protoplasts are required for RNA analysis.

CAT assay
Harvest protoplasts by centrifugation at 500 x g for 2 min. Remove the supernatant. Add 100 ml of hypotonic buffer (10 mM Tris, pH 8 and 2 mM MgCl2) and vortex vigorously for 5 sec to lyse the protoplasts. Freeze and store samples at -70oC until ready for analysis. A fast and economical xylenes extraction protocol is used for CAT assay (Seed and Sheen, 1988, Gene 67, 271-277; Sheen, in the supplement 9.6.5 of the Current Protocols in Molecular Biology, Ausubel eds). Heating the cell extract at 65oC for 10 min is unnecessary.

GUS assay
The GUS assay has been described by Jefferson (Add the cell extract into 100 ml of 1 mM MUG, incubate for 60-90 min at 37oC, add 0.9 ml 0.2 M Na2CO3 to stop the reaction, and measure the fluorescence of MU).

LUC assay
We use a Promega kit for LUC assay with either a luminometer or a scintillation counter. Use a plate reader if large number of samples are handled.

General Comments
In this system, photosynthetic gene promoters show tissue-specific and developmental (greening and normal green leaf development) regulation. In addition, regulated gene expression in response to metabolites, light, heat shock, phytohormone, redox, photooxidation, elicitor has been observed. Agents as agonists/antagonists of cellular functions and processes can be introduced into protoplasts by incubation or electroporation. Molecular genetic manipulation of cloned genes and the introduction of a dominant mutant activity can be performed. However, antisense approach is unlikely to work in transient assays. Transfection efficiency is about 30-75% with two or higher number of electroporation pulses. Co-transfection efficiency is very high (almost 100%). We have co-transfected up to 4-5 different plasmid DNA together. Transient protoplast expression assay using mutant maize leaves can also be carried out.

Solutions
Enzyme solution
1.5% cellulase R10 for green leaves and RS for etiolated or greening leaves, 0.3% macerozyme R10 (Yakult Honsha), 0.6 M mannitol, 10 mM MES, pH 5.7. Heat the enzyme solution at 50-55oC for 10 min (to inactivate proteases and accelerate enzyme solubilization) and cool it to room temperature before adding 1 mM CaCl2, 5 mM b-mercaptoethanol, and 0.1% BSA. The enzyme solution is light brown but clear. Pass the enzyme solution through a 0.45 m filter.

Electroporation, washing, and incubation solutions
0.6 M mannitol. 4 mM MES, pH5.7, and 20 mM KCl for washing and electroporation, and 4 mM KCl for incubation. Protoplasts can be incubated under light (15-50 mmol/m2s) or in the dark depending on the purpose of the experiments. About 50 (green) to 99% (etiolated and greening) of protoplasts will survive electroporation. Promega, Hoefer, BTX T-820, and modified Biorad electroporation machines work well.

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Transient Expression Assay Using Maize Mesophyll Protoplasts