RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Summary

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.

Abstract

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 behavior^6-7, 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.

Protocol

1. Transgenesis: DNA preparation

1. Dominant co-injection marker: pRL3 [Ppa-prl-1(tu92)]
   The pRL3 plasmid is a dominant co-injection marker for visually identifying successful transformation events. This plasmid encodes for a dominant mutant allele (tu92) of the Ppa-prl-1 gene closely related to the sqt-1 collagen gene in C. elegans and transforms the wildtype sinusoidal locomotion into clockwise twisting motions along the worm's body axis^1,5. The transformed animal is very similar to the popular dominant selection marker rol-6 (su1006) used in C. elegans for the ....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMI3000 Injection microscope	Leica Microsystems	DMI3000
Microinjector manipulator (Direct drive)	Tritech Research, Inc.	Narashige BC-3 Ball joint
Needle Puller	Tritech Research, Inc.	Narashige PC-10
MicroInjector™ All-Digital Multi-pressure System	Tritech Research, Inc.	Narashige MINJ-D
GeneElute™Mammalian Genomic DNA Miniprep Kit	Sigma-Aldrich	G1N70
pJet Cloning Jet kit	Fermentas	K1231
GeneJET plasmid Miniprep kit	Fermentas	K0502
DNA Clean & Concentrator™	Zymo Research Corp.	D4005
BLOCK-IT™ RNAi TOPO® transcription kit	Invitrogen	K3500-01 & K3650-01
Difco™Agar Noble	Fisher Scientific	DF0142-15-2
Microscope cover glass (1.5 - 0.16 to 0.19mm thick; Size: 50 x 45mm)	Fisher Scientific	12-554-F
Glass capillaries (filament)	A-M Systems	615000
Paraffin Oil (Heavy)	Fisher Scientific	O122-1
KH2PO4	Fisher Scientific	P386-500
Na2HPO4	Fisher Scientific	AC20651-5000
NaCl	Fisher Scientific	BP3581
MgSO4	Fisher Scientific	M80-500