I need to clone a gene of my interest in a plasmid to use for in vitro transcription into mRNA for further use in micro-injection mouse embryo for that I have to question;
what do I need in this plasmid to be suitable for invitro transcription?
second, if someone has done mRNA microinjection can tell me what is the probability of microinjection and getting proteins successfully and what to take care of while handling the mRNA and embryos in general?
Any plasmid that has a T3, T7 or SP6 promoter can be used as the commercially available IVT kits are mostly based on these promoters, the gene cloned should be flanked by a 5’UTR and 3’UTR which improves the stability of the mRNA. In our study (Reference:https://royalsocietypublishing.org/doi/full/10.1098/rsob.160190#d3e459) we used pRN3P vector which has a T3 promoter with the 5’UTR and 3’UTR derived from frog β-globin gene and T3Message Machine IVT kit was used. We used a concentration of 500ng/µl for microinjections.
The probability of the microinjection varies but we can achieve up to 90 to 95% successful microinjection and the protein expression can be monitored by tagging your gene of interest with fluorescent markers like GFP. Regarding the handling of mRNA please follow the same steps as RNA extraction is done (i.e.) RNase free working conditions. For embryos microinjection can be carried out both in M2 and KSOM medium, we each turn of injections transfer 5-7 embryos (maximum) to the cavity slide where you perform the microinjection and once done transfer the embryos to KSOM medium maintained in the CO2 incubator. Let me know if you need any other information
Good Luck
- Badges
- Science topic