I am trying to assemble TALENs for targeting a gene in zebrafish and am a
little confused about the expression vector. Bedell et al. (2012) states in
their methods section that "Once assembled, the RVDs were cloned into a
pT3TS destination vector with the appropriate TALEN backbone to generate
mRNA expression plasmid..." Does this mean I can transcribe my mRNA
directly off of a linearized Goldy plasmid after finishing Golden Gate
assembly? Or do I have to clone it into a pT3TS first? In other words, does
Goldy (specifically the RCIscript-Goldy from Addgene) make a complete mRNA
with cap, UTRs, polyA tail, etc that make it ready for injection into
zebrafish embryos? I did try to compare Goldy to pT3TS by alignment, and it
looks like Goldy is built based on pT3TS already.
I am using this RCIscript-Goldy in place of the pTAL backbone in the second
Golden Gate reaction, and the resulting plasmids look good. But I'm not
getting any cut at the target sites when assayed by PCR and restriction
digest... Am I missing something?
Without getting in any great detail, i remember there was some modifications done to improve the previous system by shortening N_ter and c_ter, a specific restriction site to differentiate from WT. Hope you took a look at suplemental data..here is a sentence where it is stated pT3TS was used for mRNA synthesis...
" GoldyTALEN scaffold was made by truncating the pTAL scaffold to 152 amino acids at the N-terminus and 63 amino acids at the C-terminus. Numerous, smaller changes have been highlighted in an amino acid comparison (Supplementary Fig. 2). The highly active pT3TS transcription factor vector was used for mRNA
synthesis..
Good luck
Thank you for your answer, Dr. Samrakandi. I guess my question is, did the authors cut the assembled TALEN repeats, along with Goldy scaffold, out of the pGoldy vector, which was used in place of pTAL vector in the second Golden Gate reaction, and put everything into pT3TS? Or did they make mRNA directly by linearizing the pGoldy vector and transcribe with T3 polymerase? My reason here is that pGoldy also has the same SacI and T3 sites as in pT3TS, and when aligned with pT3TS they look very similar. So I thought that they built Goldy on to the original pT3TS. In this case there would be no need to subclone it to pT3TS. Sorry in advance if this is confusing...
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