9

9.1　 PEG-mediated transformation

Yuji Hiwatashi

Introduction

The capacity to transform a species is a prerequisite for molecular genetic analysis.　 The transformation of P. patens has been accomplished by both the poly ethylene glycol (PEG)-mediated DNA transfer method that uses protoplast, and the particle bombardment method.　 Electroporation and gene-transfer using Agrobacterium are a matter of further experiments.　 The PEG-mediated DNA transfer method is a most reliable method for gene-targeting.　 In this chapter, the PEG-mediated DNA transfer into protoplasts is described for stable transformation.　 The transient transformation using particle bombardment is also introduced

1. Stable transformation using protoplast and PEG

Foreign DNA is introduced into protoplasts prepared from propagated protonemata.　 Regeneration rate of protoplasts is one of most critical factors for transformation efficiency.　 Protonemata grown for 3-6 days after subculture should be used for isolating protoplasts.　 Older protonemata may cause a bad transformation efficiency.

5-day-old protonemal tissue subcultured on cellophane-overlay plates

1-1 Plasmid preparation

[Materials]

QIAGEN Hi-Speed Plasmid Midi Kit, Promega Wizard Mini-prep Kit, or equivalents.

[procedure]

1. Culture E.coli harboring plasmid DNA.

2. Prepare the plasmid using QIAGEN Hi-Speed Plasmid Midi Kit or equivalent kit.

3. Digest the plasmid DNA with adequate restriction enzyme to linearize the plasmid.　

4. Purify the digested plasmid by phenol/chloroform extraction and precipitate with ethanol.

5. Dissolve the plasmid with TE, adjusting at the concentration of 0.2~1.0 µg/µl.

6. Store at –30˚C.

1-2 Transformation

We routinely transform protonemata according to a schedule below.

1. (Day 1) Start subculturing protonemata at 25˚C for about 4 (3~5) days.

2. (Day 4) Subculture the propagated protonemata at 25˚C for about 4 (3~5) days.

3. (Day 8~9) Transformation. It takes 2 days.

4. (Day 12) Incubate 3~5 days.　 Transfer top agar containing regenerating protoplasts onto a selection plate supplemented with antibiotics.　 Cultivate more than 3 weeks on the selection medium.

5. (Day 43; 3 weeks after TF) Transfer a part of colony grown onto a selection plate to antibiotic-free plate Incubate more than one week on the non-selection medium.

6. (Day 50; 4 week after TF) Transfer a part of colony grown on a non-selection plate to selection medium supplemented with antibiotics.　 Incubate more than one week.　　

7. (Day 57; 5 weeks after TF) Select lines surviving on selection medium as stable transformants.

Transformation (on the First day)

[Materials]

・ 200 ml flask x 1 (autoclave)

・ 300 ml flask x 2 (autoclave)

・ 10 ml pipet x 2 (autoclave)

・ 50 ml centrifuge tube x 2 (autoclave)

・ Funnel with a 50 µm nylon mesh (autoclave)

・ Forceps x 1 (autoclave)

・ Cellophane (autoclave)

・ Yellow for P200 and blue tips for P1000 (autoclave)

・ 0.22 µm syringe-driven filter unit and 10 ml syringe x 2

・ 0.45 µm syringe-driven filter unit and 50 ml syringe x 1

・ Neubauer hemacytometer (Becton Dickinson no. 424011)

・ Water bath x 2

・ Centrifuge

・ 50 ml conical tube (Falcon, Iwaki, etc) (sterile)

・ 15 ml conical tube (Falcon, Iwaki, etc) (sterile)

・ 15 ml conical tube (Falcon 2057) (sterile)

・ 6-cm Petri dish (sterile)

・ Parafilm

[Solution]

・ 500 ml 8% (w/v) mannitol solution (autoclave)

・ 2 g PEG6000 in a 10 ml vial and a small stir bar (autoclave)

・ 1% (w/v) MES (pH5.6)

・ 100 ml protoplast liquid medium (autoclave)　　　

H2O	90 ml
Stock A	1 ml
Stock B	1 ml
Stock C	0.1 ml
5 g/lAmmonium tartrate	1 ml (50 mg/l)
Mannitol	6.6 g (6.6%)
Glucose	0.5 g
	Fill up to 100 ml with H₂O

・ 500 ml 8% (w/v) Mannitol

・ 1 M Ca(NO₃)₂ Solution

・ 1 M MgCl₂ Solution

・ Driselase (We use driselase C-20, kindly provided by Kyowa Hakko Co., Ltd.)

[procedure]

1. Add 0.5 g of driselase in a 50 ml conical tube and then add 25 ml of 8% mannitol solution. Mix well.

2. Add 1 ml of 1 M Ca(NO₃)₂ and 100 µl of 1M Tris-HCl (pH8.0) into 9 ml of 8% (w/v) mannitol solution and mix. Filter the solution with a 0.22 µm filter.

3. (Preparation of PEG/T) Add 5 ml of the filtered solution in step (2) to the autoclaved PEG. Dissolve the PEG completely. This solution is called PEG/T.

4. (Preparation of MMM) Mix 910 mg of mannitol, 0.15 ml of MgCl₂, 1 ml of 1% MES (pH5.6) and 8.85 ml of H₂O, and then filter the solution with a 0.22 µl filter.

5. Set water baths at 45˚C and 20˚C.

6. Centrifuge the driselase solution at 4000 rpm for 5 min.

7. Transfer the supernatant to a 50 ml syringe with a 0.45 µm filter unit and filter it into a 50 ml centrifuge tube.

8. Put propagated protonema (mainly chloronema) into the driselase solution and incubate at 25˚C for 30 min.　 Mix gently every 5 min.

9. Filter the protonemata through 50 µm nylon mesh.

10. 　Centrifuge freshly isolated protoplasts at 1000 rpm (180 x g) for 2 min, and suspend gently in 40 ml of 8% (w/v) mannitol.　 Repeat this washing procedure twice.　

11. 　Count finally suspended protoplasts with hemacytometor, and re-suspend at 1.6 x 10⁶ ml^-1 in the MMM solution.

MMM (ml) = number of protoplasts per square (large nine squares) x 10⁴ (cell/ml) x 40 (ml) / (1.6 x 10⁶)

12. 　Add 30 µl of plasmid DNA into a 15 ml conical tube (Falcon 2057). Then, add 300 µl of protoplast suspension and 300 µl of PEG solution to the tube and mix gently.

13. 　Incubate the transformation mixture at 45˚C for 5 min and then at 20˚C for 10 min.

14. 　Dilute the transformation mixture by adding 300 µl of protoplast liquid medium every 3 min at 5 times and then 1 ml of protoplast liquid medium every 3 min at 5 times.

15. 　Pour the diluted protoplast solution into a 6 cm-dish and incubate at 25˚C overnight in darkness.

[Key points]

・ Suspend protoplasts in the MMM solution by pipetting gently if protoplasts aggregate on the bottom of the centrifuge tube.　

・ Use 15 ml falcon tube (2057) for transfer of plasmid.　

・ Keep temperature of a water bath at 20˚C after heat-shock.　

Transformation (on the second day)

[Materials]

・ 10 ml disposable pipet (sterile) or white tip for P5000 (autoclave)

・ Forceps x 1 (autoclave)

・ Cellophane (autoclave)

・ 15 ml conical tube (Falcon)

・ Centrifuge

・ Surgical tape

[Solution]

・ PRM/T　 Autoclave

H2O	180 ml
Stock B	2 ml
Stock C	2 ml
Stock D	2 ml
Alternative TES	0.2 ml
500mM Ammoniumtartrate	2 ml (=5 mM)
Mannitol	16 g (＝8%)
CaCl₂ 2H₂O	0.29 g (10 mM)
Agar (Sigma: A6924)	1.6 g
	Fill up to 200 ml with H₂O

・ PRM/B (1000 ml) Autoclave

H2O	900 ml
Stock B	10 ml
Stock C	10 ml
Stock D	10 ml
Alternative TES	1 ml
500 mM Ammoniumtartrate	10 ml (= 5 mM)
Mannitol	60 g (= 6%)
CaCl₂ 2H₂O	1.47 g (=10 mM)
Agar (Sigma, A6924, Nacalai Tesque: cat. no. 01028-85)	8 g (=0.8%)
	Fill up to 1000 ml with H₂O

[Procedure]

1. 　Overlay a cellophane on a 9 cm-dish containing PRM/B medium.

2. 　Remove the protoplast solution into a 15 ml conical tube (Falcon 2196) and centrifuge at 1000 rpm (180 x g) for 2min.

3. 　Re-suspend the protoplasts in 8 ml of PRM/T medium by pipetting.

4. 　Pour 2 ml of protoplast suspension on a 9 cm-dish containing PRM/B medium overlaid with a cellophane.

5. Incubate the plate at 25˚C for 3 days under continuous daylight with a light flux of 50 µmol m^-2 s^-1.　

Transfer to selection medium (4 days after TF)

[Materials]

・ Forceps x 2 (autoclave)

・ Surgical tape

[Solution]

・ Selection medium (BCDAT supplemented with adequate antibiotics)

H2O	900 ml
Stock B	10 ml
Stock C	10 ml
Stock D	10 ml
Alternative TES	1 ml
500mM Ammonium tartrate	10ml (= 5 mM)
50mM CaCl₂ 2H₂O (powder)	20 ml (= 1 mM) 　(0.15 g)
Agar (Sigma; A6924, Nacalai Tesque: cat. no. 01028-85)	8 g (= 0.8%)
	Fill up to 100 ml with H₂O

After autoclaved, allow to cool at ~50˚C and add adequate antibiotics into the medium.　 Store at 4˚C.

Antibiotics used for selection

a) Genetecin (G418)

Invitrogen (cat.no. 10131-035)　 50 mg/mL solution

Use at the final concentration of 20 mg/l

b) Hygromycin B

Invitrogen (cat.no. 10687-010)　 50 mg/ml solution

Use at the final concentration of 30 mg/l in the medium.

c) Zeocin

Invitrogen (cat.no. R250-01)　 100 mg/ml solution

Use at the final concentration of 50 mg/l in the medium.　

!NOTE!

Zeocin is light sensitive. Store zeocin solution and plates or medium containing zeocin in the dark.

d) BS-S

Funakoshi co., Ltd. (cat. no. KK-400)

Use at the final concentration of 75 mg/l in the medium.

　[Procedure]

1. 　Transfer the cellophane attached to cultures to BCDAT medium supplemented with adequate antibiotics.　 .

2. Incubated at 25˚C for 3~4 weeks under continuous light.　

　A plate after an incubation of 2 weeks

[Key points]

・ Use plates containing zeocin, which are freshly prepared.

Transfer to drug-free medium (after an incubation of 3-4 weeks on selection medium)

[Materials]

・ Forceps (autoclave)

・ BCDAT plate

・ Surgical tape

[Procedure]

1. 　Transfer each colony under antibiotic selection to antibiotic-free BCDAT medium.　

2. 　Culture at 25˚C for 1 week under continuous light.

　A plate under no selection

[Key points]

・ Transfer independent colony to the selection medium to avoid generating a chimerical colony.

・ Incubate the plate for more than 1 week.

Transfer to selection medium (after an incubation of 1 week on drug-free medium)

[Materials]

・ forceps (autoclave)

・ BCDAT plate containing adequate antibiotics

・ Surgical tape

[Procedure]

1. 　After an incubation of 1 week, re-transfer a part (protonema) of each transformant to BCDAT medium supplemented with adequate antibiotics.　 Seal the plates with a surgical tape

2. 　After an incubation of 1 week, select transformants that are able to survive on selection medium as stable transformants.

[Key points]

・ Transfer a part (as small as possible) of protonemata at the edge of a colony to selection medium.　 Do not transfer a whole colony or gametophores.

・ Use plates containing zeocin, which are freshly prepared.

・ Some kinds of disruptants may show reduced growth.　 Do not miss positive transformants even if growth of colony is reduced.

・ Note that PEG-mediated transformation will generate polyploid by protoplast fusion.　 The colony of polyploid shows different shape (typically more caulonema and few gametophore) from that of wild type.　 In this case, estimation of DNA amount using flow cytometry is needed.