Biolistic Transformation of C. elegans

Table of Contents

Table of Contents

Summary

Reagents, Materials

Plate seeding

Growing the worms

Preparation of the gold beads stock solution

DNA midi prep

Preparation of fosmid DNA coated beads for bombardment

Preparing the worms for bombardment

Bombardment procedure for PDS-1000/He (Bio-Rad)

Screening for transformants

Summary

This protocol is based on the one described in Praitis_et_al_2001, with improvements introduced in the Waterstons lab and the Hyman lab. In our hands it provides a slightly better results than the one we used previously (published in Sarov_et_al_2012) and we recommend using it instead. The protocol is adapted for use with fosmid transgenes, but it works well for all types of constructs. The protocol is for the PDS-1000/He device (Bio-Rad), which is what we use. Other devices have also been used with good success. The protocol is for a single transformation, but can be scaled up for more constructs or more rounds of the same construct, up to 4-5 transformations/day.

Reagents, Materials

Macrocarriers, Bio-Rad cat# 165-2335

Rupture Disks 1350 psi, Bio-Rad cat# 165-2329

1.5ml low retention tubes

Gold powder, Chempur 0.3-3 micron cat# 009150

1xM9 buffer

2.5M CaCl2

0.1M Spermidine, Sigma-Aldrich

50% glycerol

100% ethanol

70% ethanol

70% isopropanol

LB medium

OP50 bacteria

C600 bacteria

NGM agar plates 5 cm and 8 cm

Peptone agar plates 8 cm

M9 buffer

DNA midi prep

It’s best to isolate fosmids directly before bombardment and to only store them at 4C for up to one week. Freezing and thawing results in decline in transformation efficiency. Grow 50-500 ml cultures (depending on the number of transformations planned, 50 ml is sufficient for 2 transformation).

Standard plasmids midi or maxiprep kits can be used, but we have had variable results with different providers and you may need to check if the kit you use works well. The goal is to get pure, intact high molecular weight fosmid DNA. We try to get ~1 µg/µl final fosmid DNA concentration.  One kit that has worked reliably for us is the FosmidMAX™ DNA Purification Kit from Epicentre (FMAX046). It does not use a column, thereby minimising fragmentation and results in very good quality fosmid DNA. Pipette gently and do not vortex at any step.

Plate seeding

Grow E.coli OP50 bacteria in LB medium overnight at room temperature. Grow E.coli C600 bacteria in LB medium overnight at room temperature. Use a single colony to inoculate  200 ml media in a 500 ml flask.

Seed the plates with bacteria as follows:

Let the plates dry for 1-2 days. We use a 30°C room but on the bench should be fine too.

In addition, prepare one unseeded 10 cm NGM plate for each bombardment by drying them overnight at 37°C, and store at 4°C.

Growing the worms

1. Pick about 10 adult DP38 [unc119 (ed3)] worms on small NGM plates seeded with 100 µl OP50 and maintain at 20°C.
2. After 5-6 days DP38 worms stick together in “clumps”(L1-L3) and that’s when they are best for seeding the peptone plates. The bacteria shouldn’t be eaten up completely and there should be no dents in the agar.
3. For bombardment pick 1-2 clumps and spread the worms on 3 different spots on peptone plates seeded with C600 E.coli. DP38 don’t move well so in this way you make sure that the worms colonize the entire bacterial lawn. Use 15 peptone plates per bombardment with the hepta adapter.
4. Grow the worms for 7 days at 24.5°C. The worms are best for bombardment when there are mostly young adults on the plate. They should not be starved.

Preparation of the gold beads stock solution

The gold beads stock can be prepared in advance and kept at 4C for months. Use low retention tubes and thoroughly resuspend the beads before each use.

1. Weigh 30 mg gold powder (Chempur 009150) into a low retention 1.5 ml tube.
2. Add 1 ml 70% ethanol
3. Vortex 5 minutes
4. Soak 15 minutes
5. Spin down
6. Discard supernatant
7. Wash 3 times in 1ml sterile water
8. Resuspend in 500 µl of 50% sterile glycerol (60mg/ml final concentration)

Preparation of fosmid DNA coated beads for bombardment

Perform the coating on the day of the bombardment, using low retention tubes to minimize the DNA loss and fragmentation.

1. Wash 7 macrocarriers (Bio-Rad 165-2335) per bombardment with 100% ethanol, let dry.
2. Vortex the gold stock solution for 5 minutes
3. Label the tubes for coating.
4. Add 100µl 60mg/ml gold stock
5. Add 20 µl DNA (Maxi-prep, at least 1mg/ml)
6. Vortex 1 minute on lowest setting
7. Add 100 µl 2.5M CaCl2
8. Vortex 1 minute on the lowest setting
9. Add 60µl 0.1M spermidine
10. Vortex 30 minutes on the lowest setting
11. Let the particles settle
12. Spin down for 1 minute
13. Discard supernatant
14. Wash with 300 µl 70% ethanol
15. Discard supernatant
16. Wash with 500 µl 100% ethanol
17. Discard supernatant
18. Add 170 µl 100% ethanol
19. Mix with a pipette and distribute 20 µl to each macrocarrier.

Preparing the worms for bombardment

1. During the 30 minutes DNA binding wash the worms off the peptone plates with 1xM9 buffer into a 50 ml Falcon tube.
2. Let them settle and remove supernatant.
3. Wash once more with 1xM9.
4. Let them settle and remove the supernatant leaving no more than 2 ml.
5. Put very dry and unseeded NGM plate on ice.
6. Pipette worms onto the cooled NGM plate and leave the plate on the ice until the worms dry completely.

Bombardment procedure for PDS-1000/He (Bio-Rad)

Note: Before sample bombardment do one dry run to make sure that all the lines are drained from air and filled with helium. Please refer to the instrument manual for safety instructions.

1. Turn on the PDS-1000/He, helium and vacuum.

Dry run:

2. Dip a rupture disk in 70% isopropanol
3. Place rupture disk into the hepta adapter
4. Place the adapter into the chamber and tighten with torque wrench
5. Close chamber
6. Press the vacuum button to the upper position
7. Wait until vacuum reaches about 28 inches (26 or 27 inches work as well)
8. Press vacuum button to the lower position to hold the vacuum
9. Press the fire button
10. Release the fire button after you hear a popping noise
11. Press the vacuum button to the middle position to vent the chamber
12. Unscrew the hepta-adapter and take out the burst rupture disk

Fosmid bombardment

13. Place the DNA-loaded and dried macrocarrier into the hepta macrocarrier holder secure them with the red plastic seating tool, put on a stopping screen and a macrocarrier coverlid
14. Dip the rupture disk in 70% isopropanol
15. Place the rupture disk into the hepta adapter
16. Place the adapter into the chamber and tighten with torque wrench
17. Position the macrocarrier holder
18. Place the completely dried and cold worm plate sitting on the target shelf into the bombardment chamber.
19. Bombard the same way as described above.
20. Let the bombarded plate sit at room temperature for 1 hour
21. Wash worms with approximately 4ml 1xM9 off the plate
22. Spread the washed off worms evenly onto 20 large NGM plates
23. Let the worms grow at 24.5°C for 2 weeks, start screening.

Screening for transformants

Start screening after 2 weeks, at which point a good line should have a lot of unc rescued worms moving around the plate. Movers that appear earlier are often unstable array lines, not worth picking.

1. Pick several worms from each rescued plate (4-6) and move them to 4cm NGM plates seeded with OP50
2. Check in the next generation for uncs. Keep the lines with 100% unc rescued progeny as likely integrants. Some worms may be heterozygous integrants and will segregate ~25% unc. Keep picking rescued worms for several generations - if you get a 100% rescued progeny keep the line.
3. Inspect the lines for GFP fluorescence. heMost fosmid transgenes will express at endogenous levels, which for most genes will only be visible on a high magnification compound microscope, but not on most fluorescent dissection microscopes. Scoop 20-30 worms from a mixed stage plate and mount them in a drop of M9+5mM levamisole. Inspect multiple worms of various stages. Lines that do not express have likely integrated only a part of a fragmented transgene. Lines that appear mosaic are likely arrays. However for genes with unknown expression be more careful - the gene may be expressed in a specific developmental stage or environmental conditions.
4. Maintain the expressing integrants for 3 generations and check for 100% transmission. 100% transmission does not guarantee with certainty that the line is stable/integrated but is a good rule of thumb.
5. If necessary, clean the lines by bleaching and freeze stocks.
6. Be cautious with any transgenic line, and if possible compare multiple independent lines. If possible validate the transgenes with genetic rescue experiments.