Help with shRNAmiR30 based constructs (for a simple MD) - (Sep/14/2013 )
Hello fellow scientists;
I am currently trying to make my own miR30 based shRNA according to Dow et al. with a 97-mer template and a PCR reaction to create a product which I want to clone in a specific vector (f.e. TRIPZ or pMSCV-FLIP). What I do not understand is how the miRshRNA is exactly transcribed; in the vector maps it looks like it doesn't contain a start codon nor does SNAPGENE recognise any specific promoter; it looks like you insert the 110bp shRNAmiR after GFP/RFP directly (with a small stretch of DNA in between). Can anybody explain to me how this exactly works; is enough to just insert my 110bp sequence (containing shRNAs, loops etc) flanked by XHO1/ECOR1 (for pMSCV-FLIP) or by XHO1/MLU1 (For Tripz) directly?! I hope the question is clear and help this MD who got lost in biomedical research...
So you want to clone a shRNA not a miRNA, right? Yes, TRIPZ is for cloning shRNA. In this vector, the inducible promoter TRE drives the expression of RFP + shRNA as one transcription unit. The start and stop codons should be at the beginning and end of the RFP, respectively. After the transcription unit is transcribed into mRNA, the RFP ORF will be translated into RFP protein, the 3UTR containing the shRNA will be cut and processed into shRNA. You don't need to worry about start codon and other elements in the vector. According to the manual, it seems the MCS is between XhO1 and MLU1, it is where your shRNA should be inserted. Have you already cloned your shRNA in some other vector as suggested by the Thermo manual so that you can cut the insert using xho1/mluI and subclone it into TRIPZ?
As for pMSCV-FLIP, it is for cloning miRNA and uses ligation independent cloning method, which means you have to look at the vector manual to learn how primers should be designed to amplify your insert. After the insert is amplified by PCR, you anneal the insert and specially prepared vector to let the insert incorporate into the vector.
Thanks for the quick and clear answer. As is understand from the pTRIPZ manual the shRNA used in this vector are also based on miR30.
I have some nicely working shRNAs that I wanted to translate and clone in shRNAmiR30 format. In the TRIPz manual
they describe subcloning from their other vectors (pGIPZ f.e.), however they do not describe a protocol for designing your own
shRNAmiR. I designed a 97-mer oligo and two primers with the Xho1 and Mlu to amplify the shRNA and subsequently cut with
these enzymes and clone the 110bp product (the shRNAmiR as descibed in the article I mentioned) into the XhoI/Mlu1 site
of pTRIPZ. I was just wondering if this was enough or that I needed to add other sequences (promoters/LTRs etc), since the
cloning product described in the TRIPZ manual is 354bp, but as I understand from you this will not be necessary since it is
just part of the 3'UTR like endogenous miRs. I still do not fully understand the term ligation independent cloning since you need
to ligate your PCR product into your destination vector anyhow, but that is just a matter of semantics I suppose. Thanks a lot and please
correct me if I misunderstood your comments.
PS so if I understand correctly a 'normal' shRNA would also do the job?
It seems that the cloning of shRNA into pTRIPZ is not that straightforward if you don't have pGIPZ vector.
The 354 bp fragment released by XhoI/MluI from pGIPZ contains the shRNA core sequence flanked by precursor sequences that also flank miR-30. Unless you know there are some convenient restriction size flanking the shRNA core, you need to have the pGIPZ vector so that you can take advantage of the miR-30 structure based pTRIPZ vector which is supposedly give you high knockdown efficiency. If there are restriction sites available in the pGIPZ vector allowing to just insert the shRNA core which is about 90 bp as you mentioned, you can just order two strand of DNA oligos. The oligos should be designed in a way, when annealed, give you the desired restriction digested ends. You don't need to synthesize oligos, anneal them and then amplify using additional primers.
I believe Thermo intentionally did not include any cloning site around the shRNA core to force people to buy the other vector from them.
The original paper from Hannon group reporting the miR-30 based shRNA expression method should be helpful http://cshprotocols.cshlp.org/content/2013/7/pdb.prot075853.full
ligation independent cloning applies only to pMSCV-FLIP. you can search "ligation independent cloning" to see what it means.