Plasmid DNA containing a functional leech promoter and in vitro transcribed mRNA are the two available options for expressing molecular constructs in Helobdella embryos.

Preparation of DNA

pBSMNEF1P is a plasmid backbone that can drive transgene expression in Helobdella and possibly other annelid species (Gline et al., 2009). A new plasmid backbone (pMiEF1P) that allows genome integration by transposase is now available for use. Plasmid DNA is best prepared using kits that yield transfection-grade DNA, e.g. Qiagen Plasmid MIDI Kits (Cat. Nos. 12243, 12245, 12643, 12943, 12945) or equivalents. We found ZymoResearch ZR Plasmid Miniprep Kit (Cat. Nos. D4015, D4016, D4054) a useful option for transfection-grade small-scale miniprep. Plasmid DNA for injection must be dissolved in nuclease-free water (DO NOT use TE buffer; EDTA interferes with normal cellular physiology!). Store transfection-grade plasmid DNA in -20˚C or -80˚C.

Preparation of RNA

We routinely use pCS2 and pCS107 as our base vector backbone for building plasmid constructs for mRNA synthesis. mRNA is transcribed from linearized plasmid or PCR product using mMESSAGE mMACHINE SP6 Kit (Ambion AM1340) [T7 (AM1344) or T3 (AM1348) kits may be used for other vector backbones]. If PCR has to be used, use high-accuracy proof-reading enzymes such as Phusion (Finnzyme F-530) to minimize PCR-induced mutation in your template. We used a specially designed primer pair for pCS for optimal performance using Phusion, and they are modified from SP6 and T3 primers. Performing in vitro transcription following manufacturer's instruction. We usually allow transcription reaction to go overnight. In the following day, an optional polyadenylation reaction is performed using a Poly(A) Tailing Kit (Ambion AM 1350; this kit is designed for used with Ambion mMESSAGE mMACHINE kits). Artificially poly(A)ed mRNA has performed much better than synthesized mRNA without poly(A)ed.

To save time and to improve yield and purity, we routinely use a spin column to purify synthesized, poly(A)ed mRNA. We have been using ZymoResearch RNA Clean & Concentrator-5 (Cat. Nos. R1015, R1016; capacity: up to 10 μg of RNA for each column) or -25 (Cat. Nos. R1017, R1018; capacity: up to 50 μg of RNA each column). When using R1015/R1016, we divide a single reaction into halves and load each half into a column (i.e. two columns are used for each 20 μl transcription reaction (or 100 μl poly(A) reaction) and elute RNA with 6-15 μl of nuclease-free water. But, if you are looking for a more economical option, LiCl precipitation of synthesized mRNA has been working pretty well. After washing and drying the RNA pellet, dissolve mRNA in nuclease-free water. For a standard 20-μl reaction, we usually dissolve RNA in 6 μl of nuclease-free water.

Take 1 μl of RNA stock and dilute it further for quality analysis and quantification. After A260 quantification, adjust RNA concentration (to a concentration that is convenient for handling) by adding an appropriate amount of nuclease-free water. Aliquot the RNA into 0.5 μl/tube. Store them in -80˚C. Before injection, add an appropriate amount of monitor dye to a 0.5 μl RNA aliquot (1 μl of phenol red or phenol red + fluorescent dextran, if you want to inject RNA at 1 μM and your RNA stock concentration is 3 μM). There is no need to be super paranoid about RNase (no need to DEPC everything), but RNase contamination could still a problem. Just try to do your best to avoid RNase contamination. For example, use a filter pipette tip for handling RNA, use nuclease-free reagents and tubes for RNA synthesis and storage, do not contact the ends of capillary tubing (for making micropipettes) with bare skin, and wear gloves when pulling and handling micropipettes.

Injection

Optimization of DNA or RNA concentration is required for each experiment. A good starting point is 100 ng/μl (in micropipette) for plasmid DNA and 1 μM or 1 μg/μl (in micropipette) for mRNA. [The estimated volume of routine teloblast injection is about 2-3 picoliter (1:100 of cell volume). For zygote injection, the volume is about 50 picoliter (1:1000 of cell volume).] We found that Fast Green has a negative effect on the expression of injected DNA or RNA. We use phenol red solution (Sigma P0290) as a monitor dye when injecting DNA or RNA. Fluorescent dextran can be mixed into phenol red to follow the development of the injected cell.