TITLE: Transposase injection mix protocol
AUTHORS: Ben Matthews

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details the steps necessary to generate injection mixes (comprised of plasmid DNA for integration and transposase mRNA) for transposon-mediated integration into </span><span style = "font-style:italic;">Aedes aegypti</span><span> and other insects. It has been empirically tested in </span><span style = "font-style:italic;">Ae. aegypti</span><span> embryos with piggyBac transposase. In our hands, using mRNA as a source of transposase instead of a helper plasmid increases transformation rates substantially.</span></div></div>

[STEPS]
?. [Purifying injection-ready integration plasmid]
Perform a midiprep of the integration plasmid from a  overnight culture of your transposon integration plasmid.
50 ml
?. [Purifying injection-ready integration plasmid]
Follow the kit protocol (if using recommended kit: Machery Nagel Nucleobond EF)
Be sure to follow the instructions precisely for all endotoxin-removal steps.
?. [Purifying injection-ready integration plasmid]
Fully dry the final pellet, and re-suspend in , with nuclease free pipettes and tips.
[nuclease free water]
?. [Purifying injection-ready integration plasmid]
Quantitate by NanoDrop or Qubit and record final concentration.
Should be > 1 μg/μL - if much higher, should be diluted with nuclease free water to ~2 μg/μL and re-quantified.
?. [Purifying injection-ready integration plasmid]
Aliquot and store at  (unless preparing injection mix same-day, in which case, plasmid can be left on ice).
-80 °C
If using the kit recommended here, plasmid is injection ready and needs no additional cleanup (at least for Ae. aegypti).
?. [Generating transposase mRNA by in vitro transcription: template PCR]
PCR: To generate DNA template for IVT, perform PCR from a plasmid containing transposase cDNA. The primer sequences below represent an amplification strategy for wild-type or a hyperactive form of piggyBac transposase. To generate template for a different transposase (eg Mos), add the T7 polymerase initiation sequences and a linker to the 5’ end of a forward primer corresponding to the first 20-30 bp of the transposase sequence. Use an appropriate primer to match the reverse complement of the last 20-30 bp of the transposase sequence. 
 pBac forward primer (bold/italic represents T7 initiation sequence and necessary linkers):            GAAACTAATACGACTCACTATAGGGAGAGCCGCCACATGGGTAGTTCTTTAGACGATG
 pBac reverse primer (wild-type PBac, our plasmid obtained from ITF/Rob Harrell): 
            CTTATTAGTCAGTCAGAAACAAC 
 Alternate pBac reverse primer:            TCAGAAACAACTTTGGCACATATCA This reverse primer can be used when amplifying cDNA for a hyperactive PBac, described in Otte et al., 2018.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Perform a PCR reaction (scaled to 100 μL), using ~1 ng plasmid as template.
I use KOD, but any polymerase should work. Adjust time and temperature accordingly. For KOD, I used an annealing temperature of 56° and extension time of 40s.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Run an agarose gel to verify the presence and appropriate size of the template.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
To cleanup product, perform magnetic bead purification with RNAClean XP beads as described in the following sub-steps.
All following steps should be performed with nuclease free water/tips/tubes/pipettes!!
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Combine 1.5x volume of RNAClean XP beads to the PCR reaction in a 1.5 ml eppendorf tube.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Vortex to mix well.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Incubate for  at .
0 Room temperature
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Place on magnet stand until solution is clear; approximately .
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Remove supernatant.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Rinse beads with . (1/2)
[freshly made 80% EtOH]
Use nuclease-free alcohol and water!
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Let stand for -. (1/2)
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Remove supernatant. (1/2)
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Rinse beads with . (2/2)
[freshly made 80% EtOH]
Use nuclease-free alcohol and water!
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Let stand for -. (2/2)
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Remove all remaining ethanol - switch to 10 μL tip! - and air dry for  until pellet is completely dry.
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Remove from magnet stand and re-suspend bead pellet in .
[nuclease free water]
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Incubate  at  and then return to magnet stand.
0 Room temperature
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Once solution has fully cleared (~), carefully transfer  with a 10 μL tip to a separate tube.
[supernatant]
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Check concentration with a spectrophotometer (Nanodrop) or Qubit.
Hopefully >250 ng/μL!
?. [Generating transposase mRNA by in vitro transcription: template PCR]
Proceed to in vitro transcription (IVT) - any leftover template can be stored at  and used for subsequent IVT reactions.
-20 °C
?. [Generating transposase mRNA by in vitro transcription: IVT]
Perform 20 μL IVT reaction per NEB protocol (briefly descipbed in the following steps).
All following steps should be performed with nuclease free water/tips/tubes/pipettes!!
?. [Generating transposase mRNA by in vitro transcription: IVT]
Add reagents in the following order.
?. [Generating transposase mRNA by in vitro transcription: IVT]
Nuclease free water (to 20 μL total reaction volume)
?. [Generating transposase mRNA by in vitro transcription: IVT]
[2X ARCA/NTP Mix]
?. [Generating transposase mRNA by in vitro transcription: IVT]
[PCR template ]
2 μL - should be 500-1000 ng
?. [Generating transposase mRNA by in vitro transcription: IVT]
[T7 RNA Polymerase Mix]
?. [Generating transposase mRNA by in vitro transcription: IVT]
Mix thoroughly and pulse-spin to collect.
?. [Generating transposase mRNA by in vitro transcription: IVT]
Incubate for  at  .
37 °C
?. [Generating transposase mRNA by in vitro transcription: IVT]
Add .
[DNAse I]
?. [Generating transposase mRNA by in vitro transcription: IVT]
Remove  and save on ice for sizing and integrity analysis.
1 µl
?. [Generating transposase mRNA by in vitro transcription: IVT]
Add the following reagents.
?. [Generating transposase mRNA by in vitro transcription: IVT]
[nuclease free water]
?. [Generating transposase mRNA by in vitro transcription: IVT]
[10x Poly(A) polymerase reaction buffer]
?. [Generating transposase mRNA by in vitro transcription: IVT]
[Poly(A) polymerase]
?. [Generating transposase mRNA by in vitro transcription: IVT]
Incubate for  at  .
37 °C
?. [Generating transposase mRNA by in vitro transcription: IVT]
Proceed immediately to cleanup and validation.
?. [mRNA cleanup]
Cleanup in vitro transcription reaction with RNAClean XP beads as described in the following substeps.
?. [mRNA cleanup]
Combine 1.5x volume of RNAClean XP beads to the PCR reaction in a 1.5 ml eppendorf tube.
?. [mRNA cleanup]
Vortex to mix well.
?. [mRNA cleanup]
Incubate for  at .
0 Room temperature
?. [mRNA cleanup]
Place on magnet stand until solution is clear; approximately .
?. [mRNA cleanup]
Remove supernatant.
?. [mRNA cleanup]
Rinse beads with . (1/2)
[freshly made 80% EtOH]
Use nuclease-free alcohol and water!
?. [mRNA cleanup]
Let stand for -. (1/2)
?. [mRNA cleanup]
Remove supernatant.
?. [mRNA cleanup]
Rinse beads with . (2/2)
[freshly made 80% EtOH]
Use nuclease-free alcohol and water!
?. [mRNA cleanup]
Let stand for -. (2/2)
?. [mRNA cleanup]
Remove all remaining ethanol - switch to 10 μL tip! - and air dry for  until pellet is completely dry.
?. [mRNA cleanup]
Remove from magnet stand and re-suspend bead pellet in .
[nuclease free water]
?. [mRNA cleanup]
Incubate  at  and then return to magnet stand.
0 Room temperature
?. [mRNA quantitation and sizing verification]
Quantitate using spectrophotometer (Nanodrop) or Qubit.
A successful IVT reaction should generate 50 μL of purified product at >400 ng/μL.
?. [mRNA cleanup]
Once solution has fully cleared (~), carefully transfer  with a 10 μL tip to a separate tube.
[supernatant]
?. [mRNA quantitation and sizing verification]
Run  (alongside ) on a Bioanalyzer, Tapestation, or agarose gel with an RNA ladder.
[final mix]
[reserved pre-poly(A)-tailing]
?. [mRNA quantitation and sizing verification]
Things to look for:Pre-poly(A)-tailing: a single, bright band with no sign of degradation productsPost-poly(A)-tailing: one or multiple bands, broader in size distribution but all product should be longer than the pre-tailing product
This procedure should generate >20 μg of injection-ready mRNA.
?. [mRNA quantitation and sizing verification]
Proceed immediately to final injection mix preparation. Any leftover mRNA should be aliquoted and stored at .
-80 °C
?. [Final injection mix preparation]
Combine mRNA and donor plasmid at final concentrations of 300 ng/μL each (or any other desired concentration). Any necessary dilution must be done with nuclease free water or nuclease free injection buffer, as appropriate. For Ae. aegypti, we use water.
?. [Final injection mix preparation]
Aliquot injection mix into  aliquots in small nuclease-free tubes.
5 µl
?. [Final injection mix preparation]
Freeze at .
-80 °C
?. [Final injection mix preparation]
Keep injection mixes frozen until immediately before injection. Thaw a single aliquot on ice for an injection session, and do not re-freeze.
?. [Generating transposase mRNA by in vitro transcription: IVT]
Mix thoroughly by pipetting up and down.
?. [Generating transposase mRNA by in vitro transcription: IVT]
Incubate for  at .
37 °C