In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.
Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals’ own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.
1. Transgenesis: DNA preparation
pRL3 | 4 μg |
10xbuffer | 10 μl |
PstI (10 U/μl) | 4 μl |
dH2O | ~ |
Total: | 100 μL |
Ppa-egl-4p::gfp | 4 μg |
10x buffer | 10 μl |
SalI (10 U/μl) | 4 μl |
dH2O | ~ |
Total: | 100 μl |
gDNA PS312 | 10 μg |
10x buffer | 10 μl |
Pst I and Sal I (10 U/μl) | 8 μl |
dH2O | ~ |
Total: | 100 μl |
Table 1. Mixture for Restriction Enzyme Digest.
Genetic material | Concentration |
Ppa-egl-4p::gfp (SalI) | >5 ng/μl (0.1-10ng/μl) |
pRL3 (PstI) | >1 ng/μl |
gDNA PS312 (PstI + SalI) | >60 ng/μl |
dH2O | ~ |
Total: | 30 μl |
Table 2. Ppa-prl-1 injection mixture
2. RNA Interference: In vitro double stranded RNA synthesis
3. Microinjection: Protocol for injection of transgenes and/or dsRNA
Figure 1. A schematic drawing of the P. pacificus anatomy. The clear nucleated cells are female germ cells (oocytes) and the gray shaded part is the intestine. Only one distal anterior gonad is shown but notice the two distal gonads cross each other dorsally near mid-body.
Figure 2. Images of microinjection. (A) The injection needle tip is in focus with the far right line of the pulled capillary piece. By lightly touching the tip of the needle to that edge, the needle tip should break while still retaining a sharp point. (B) The needle inserts into the top gonad just above the gut curvature for the first injection. (C) The needle inserts into the bottom gonad just below the gut curvature for the second injection.
4. Representative results:
1. Result of Transgenesis
Session | Injected P0 | F1 Rollers | % transgenic F2 | Average % transmission after F2 |
1 | 60 | 2 | (line 1) 0% (line 2) 13% |
NA 13%±10 |
2 | 40 | 1* | (line 3) 0% | NA |
3 | 40 | 0 | 0% | NA |
4 | 40 | 1 | (line 4) 0% | NA |
5 | 20 | 0 | 0% | NA |
6 | 40 | 1 | (line 5) 26% | 22%±10 |
* a male roller that did not cross; NA: Not applicable
Table 3. Results of injected PS312 with Ppa-egl-4p::gfp (Sal I digested), pRL3(roller) (Pst I digested) , and gDNA PS312 (PstI and SalI digested).
Figure 3. (A) wildtype (B, C) A stable F4 pRL3; Ppa-egl-4p::gfp transgenic line showing head neuron GFP expression.
2. Result of RNA interference
Figure 4. RNA injected prl-1 mutants show complete knockdown of “rolling” locomotion. (A) prl-1 roller gain-of-function mutant tu92. (B) “knocked down” prl-1 mutant exhibit normal wildtype posture and locomotion. (C) Injected prl-1 (bottom) also has longer body than prl-1 mutant (top). (D) The longer body of injected prl-1 (bottom) is also longer than the wildtype PS312 (top). The longer body phenotype was also observed in the rol-5 (sqt-1) RNAi knockdown of C. elegans N2 (data not shown).
roll | non-roll | |
A prl-1 dsRNA (200 ng/μl) | 13 | 21 |
B prl-1 dsRNA (1000 ng/μl) | 9 | 16 |
control | 20 | 2 |
Table 4. Summary of Ppa-prl-1 dsRNA injections. (A) [dsRNA] = 200 ng/μl; (B) [dsRNA] = 1000 ng/μl. P = 0.0019 and 0.041 by Fisher’s Exact Test, two-tailed, for 200 and 1000 ng/μl injections, respectively.
P. pacificus populations are found in close association with various scarab beetle species worldwide and is a model nematode intermediate between free living and parasitic nematodes. The strength of the P. pacificus as an emerging model organism lay in the integration of its genetic and physical maps that promote positional mapping of mutants isolated from unbiased forward genetic screens (i.e. not just for candidate genes previously characterized in C. elegans)6,10. However, C. elegans genetic techniques are not readily transferable to P. pacificus due to the significant differences in organ morphology, primary DNA sequences, as well as response to foreign DNA. Our present study illustrates in detail how to introduce stable reporter genes previously described by Schlager et al5 and how to knock down the same dominant roller mutant by RNAi. Our protocol does not presume prior knowledge of transgenesis or RNAi in C. elegans.
The F1 transformation rate shown in this study (˜2%) is comparable to previous results using the prl-1 marker in P. pacificus (2-10%)5, but less than the rate found in S. stercoralis (3-22%)4. There is still much potential for improvement of transgenic technology in P. pacificus. Even using the prl-1(tu92) dominant roller marker homologous to the commonly used rol-6 (su1006) allele in C. elegans, the effectiveness of transgenesis in P. pacificus is significantly less than those first described for C. elegans by Mello and co-workers3, in which multiple transgenic F1 progeny can be obtained per injected animal in expert hands. Several factors may explain this difference: (1) The lower number of oocytes in diakinesis in the P. pacificus female germline (average 1 per gonad)8 may reflect a lower rate of mitotic germ cells transitioning to meiosis in P. pacificus compared to C. elegans11. Hence, fewer oocytes can take up DNA and RNA following each injection. The overall lower brood size per hermaphrodite in P. pacificus PS312 (˜200) compared C. elegans N2 (˜300) may also exacerbate the lower number of the transgenic F1 per injected animal. (2) The requirement for genomic DNA in the injection mix for transmission of foreign DNA from F1 to F2 suggest a stronger gene silencing mechanism may also be involved in P. pacificus than in C. elegans. Nevertheless, P. pacificus transgene expression does not seem to undergo the extreme gene silencing found in S. stercoralis in which transgene expression is limited to F1 animals4. We are currently characterizing the expression of PPa-egl-4p::gfp lines in F6 animals. One straightforward method to improve rates of transgenesis is to increasing the concentration of the roller co-injection marker (currently 1 ng/μl of pRL3 compared to 25-50 ng/μl of pRF4 in C. elegans).
The ability to manipulate gene function at the organismal level by RNAi and transgenesis are the twin pillars of technology that elevate C. elegans above many other model organisms. We hope our current study will greatly enhance the P. pacificus model for genetics studies in development and behavior by providing easily accessible instructions for transgenesis and RNAi.
The authors have nothing to disclose.
The authors are very grateful to RJ Sommer and X Wang for assistance with microinjection, as well as insightful comments from the anonymous reviewers. This work is supported by NIH grant SC2GM089602.
Product: | Catalog #: | Supplier: |
---|---|---|
DMI3000 Injection microscope | DMI3000 | Leica |
Microinjector manipulator (Direct drive) | Narashige BC-3 Ball joint | Tritech Research |
Needle Puller | Narashige PC-10 | Tritech Research |
MicroInjector™ All-Digital Multi-pressure System | Narashige MINJ-D | Tritech Research |
GeneElute™Mammalian Genomic DNA Miniprep Kit | G1N70 | Sigma |
pJet Cloning Jet kit | K1231 | Fermentas |
GeneJET plasmid Miniprep kit | K0502 | Fermentas |
DNA Clean & Concentrator™ | D4005 | ZYMO Research |
BLOCK-IT™ RNAi TOPO® transcription kit | K3500-01 & K3650-01 | Invitrogen |
Difco™Agar Noble | DF0142-15-2 | Fisher Scientifi |
Microscope cover glass (1.5 – 0.16 to 0.19mm thick; Size: 50 x 45mm) | 12-554-F | Fisher Scientific |
Glass capillaries (filament) | 615000 | A-M systems |
Paraffin Oil (Heavy) | O122-1 | Fisher Scientific |
KH2PO4 | P386-500 | Fisher Scientific |
Na2HPO4 | AC20651-5000 | Fisher Scientific |
NaCl | BP3581 | Fisher Scientific |
MgSO4 | M80-500 | Fisher Scientific |