Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.
The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.
Overview
Traditionally transgenic worms were generated by microinjecting transgene DNA into the C. elegans germline 1,2,3,4. While successful, this approach required specialized equipment and the development of experience with worm anatomy and the microinjection technique. More recently biolistic bombardment has been developed as an alternate approach which uses DNA-coated gold particles to introduce foreign DNA into the germline 5,6. This approach requires less time investment in terms of practice to become successful.
The biolistic bombardment of C. elegans to generate transgenic worms has low efficiency at the level of the individual worm so success requires a large quantity of animals. These can be grown in several ways, but we use egg plates to reduce the work and number of plates involved. Our protocol starts with the preparation of egg plates and worms and then focuses on the bombardment protocol using the Bio-Rad PDS-1000/He Hepta System. We adapted our protocol in part from work of Berezikov et al 7.
With bombardment, the unc-119 gene is typically used as a marker for transgenic animals. Worm strains carrying this mutation are severely uncoordinated and barely move, are dumpy in appearance, and are unable to form dauer larvae 8. The dauer is an alternate larval stage which is used to delay development to a reproductive adult under adverse conditions, and the entry into dauer is regulated by multiple genes 9. Several strains carrying the unc-119 gene are available from the C. elegans Genetics Center (CGC, Minneapolis, MN). The original DP38 strain (unc-119(ed3)) also carries an unrelated dauer-formation constitutive (daf-c) mutation which could affect downstream experiments. Several labs have outcrossed this mutation, and the HT1593 strain is available from the CGC. We find that transgenic animals are somewhat easier to identify with the DP38 strain and tend to use this strain for creating transgenic animals. When necessary, we use HT1593 to outcross the transgenic worms to remove the daf-c mutation. Getting the DP38 worms to grow is one of the slowest steps in the protocol so we maintain a stock of growing plates while preparing the transgenes.
Expression of the unc-119 marker along with the gene of interest allows easy identification of the animals expressing the transgene based on rescue of normal motility and body size 5. The unc-119 gene can be obtained from several sources, and vectors using the unc-119 genomic DNA, unc-119 promoter and cDNA, and the genomic DNA for the smaller C. briggsiae gene are used 8,10. We recently described a simple protocol to add an unc-119 cassette to any plasmid containing an ampicillin resistance gene by homologous recombination 11 and a method to modify worm fosmids to carry the unc-119 gene by Cre-lox recombination to generate transgenic animals 12. The plasmids are available from Addgene Inc. (Cambridge, MA).
While the effort involved in performing a single bombardment is significant but manageable, the additional work involved in performing multiple bombardments on a single day is minimal. Consequently, we routinely cluster the production of transgenic worms to decrease the work involved in generating each.
1. Egg Plate Preparation
The unc-119 mutant worms are difficult to grow on standard NGM plates because with their impaired mobility they tend to starve on parts of a plate while other parts of the plate still contain food. Egg plates solve this problem as the thick food layer on egg plates allows unc-119 worms to crawl more easily and to take over all the plate 7. The egg plates also have the benefit of supporting the growth of a large number of worms so fewer plates are needed 13. Usually 5 egg plates are sufficient to grow worms for one bombardment. The recipe below makes roughly 50 plates but can be halved to make fewer if desired.
The egg plates can become easily contaminated so we use both antibiotics and antifungal drugs in the plates and only use eggs generated by hypochlorite treatment for seeding the plates.
Day 1:
Day 2:
Day 3:
Day 4:
2. Seeding Egg Plates
Bombardment
We prepare the gold stock ahead of time and keep it stored at 4°C for use. Also needed are unspotted and spotted 10 cm NGA plates. The unspotted plates need to be poured well in advance and allowed to thoroughly dry to facilitate absorption of the liquid added with the worms.
On the day of the bombardment, we wash off the worms first then start preparing the DNA-coated gold particles. We then add the worms to the unspotted NGA plates and finish washing the gold particles and transferring them to the macrocarriers.
Gold particle preparation:
Worms:
This step is important: the plate should be dry and the worms evenly dispersed on the NGA plate. Keeping the worms on ice prevents them from moving on the plate and aggregating into piles.
DNA preparation (for one bombardment):
Preparation of macrocarriers:
3. Bombardment
Be sure to perform a blank bombardment before the experiment to flush helium through the system.
Worm recovery after bombardment:
4. Representative Results
The success of the protocol with regards to obtaining transgenic animals depends on the particular transgene. For promoter:GFP reporter transgenes we have obtained up to 20 lines. A more typical result is 3-10 lines. Up to 30% of the transgenic lines are integrated lines, but this is random and we will perform multiple bombardments on a single day if an integrated line is particularly desired.
Biolistic bombardment is a simple method to introduce foreign DNA into many organisms, including C. elegans 1,5,6,7,16. It relies upon gold particles forming a complex with DNA in the presence of CaCl2. Cationic polyamines, such as spermidine, protect DNA from nuclease degradation in vivo. Since spermidine is a labile molecule, it is important to store it in small aliquots at -20°C, and make the solution right before performing the bombardment. As we described in the bombardment protocol, protamine can be used instead of spermidine to deliver foreign DNA 15. Protamine has the advantage of being more stable at room temperature and being a powder instead of a viscous liquid.
The unc-119 rescue gene can be placed either in cis on the same plasmid as the transgene or on a separate plasmid that is mixed with the transgene prior to coating the gold particles. While the mixing of one or more plasmids is usually successful, we and others have found that bombardment of worms with multiple plasmids can create transgenic animals carrying some, but not all the plasmids 6,11. To facilitate placing the unc-119 gene on the transgene plasmid, we recently described a protocol using homologous recombination to insert the unc-119 gene into the ampicillin resistance gene of almost all plasmids 11.
Further, to facilitate moving transgenes from the DP38 strain into worm strains lacking the unc-119 mutation, we also generated a plasmid with unc-119 fused to mCherry 11. The pan-neuronal mCherry fluorescence can then be used to track the presence of the transgene in a variety of genetic backgrounds 11.
Some transgenes might be difficult to detect after bombardment due to weak expression or a stage specific expression. The presence of the transgene can be verified often via the use of PCR using the transgenic worms. For transgenes with weak expression, performing additional bombardments can lead to the identification of lines with stronger expression. Alternately, the use of a compound or confocal microscope can often help with visualizing the expression of fluorescent proteins.
The authors have nothing to disclose.
This work was supported by seed funds from the University of Pittsburgh and NIH grant AG028977 to A.L.F.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Gold particles | Inbio gold | BD021 | 1micron | |
Midiprep kit | Qiagen | 12143 | ||
Spermidine | Sigma | S4139 | ||
Protamine | Sigma | P4505 | ||
Macrocarriers | Biorad | 165-2335 | ||
PDS-1000/He Hepta System | Biorad | 165-2257 | ||
Rupture disk | Biorad | 165-2330 | ||
Nystatin | Sigma | N1638 | ||
Streptomycin | MP | 100556 |