Hello everyone.
I am trying to construct a lentiviral construct for inducible expression of long-noncoding RNAs (lncRNA) linked to the expression of fluorescent protein ( co-expression from a single vector, not physical link/fusion, in a doxycycline-dependent manner).
I am trying to achieve the following:
- Transduce the cells
- Check for baseline expression (tightness/leakage of TRE promoter) by qPCR/FACS
- Treat cells with Dox
- Sort uniform population (mean fluorescence) of cells by FACS
- Withhold dox
- Check for reversibility of expression by qPCR/FACS
- If necessary, sort for fluorescence-negative cells without the presence of Dox
- Correlate the expression (Dox response curve) of lncRNA(qPCR) and mean fluorescence and use fluorescence as a proxy in downstream experiments where exact expression measured by qPCR is not necessary. (Possibly also to quantify % of cells that are being induced and their induction efficiency in an animal model -extracted tumor tissue)
lncRNAs (unlike protein-coding genes) are not translated and carry their function directly upon transcription as RNA transcripts. It is thus important that following transcription, the length/ structure of lncRNA transcript is not altered by its fusion with RFP. This is not an issue for protein-coding genes, where IRES and P2A (and other self-cleaving peptides) can be utilized to obtain separate proteins upon translation.
I have considered following strategies. I will appreciate any insight you can provide on those, as well as any alternatives you may suggest.
Two separate inducible promoters:
TRE>lncRNA.... TRE>Flourescent protein. I think this strategy is cleaner than the alternative, however, I have been advised against, due to possible recombination between TRE promoters. Is this really an if I am using Stbl3 or other recA mutant strain for cloning of my lncRNA of interest? Can the vector be modified in a way that Amp/Kan selection will prevent recombinant clones from propagating?
Transcriptional polycistron - Inspired by following article:Article Polycistronic tRNA and CRISPR guide-RNA enables highly effic...
The idea here is that a single TRE promoter could drive the expression of both lncRNA and fluorescent protein. The inclusion of tRNA in between would result in RNA cleavage and both transcripts could go their separate ways. I see several concerns with this strategy, however.
TRE>lncRNA|tRNA|FP:polyA. In this arrangement, the FP part of the transcript would be missing the 5' cap, likely influencing its stability and translation. I was thinking if the inclusion of IRES could do the trick here? TRE>lncRNA|tRNA|IRES:FP:polyA.
In the reverse arrangement, TRE>FP|tRNA|FP:polyA, the FP part of the transcript would be missing the polyA, likewise influencing the stability/translation. I do not know how could this be improved.
My second major concern is that tRNA cleavage will not be efficacious enough, and I will end up with a high proportion of full length transcript (lncRNA+FP) floating around, which can bias the experiments. I can measure this experimentally, but not before I get my hands on such constructs. Do you think that including several tRNAs could facilitate more efficacious cleavage?
I am clearly out of my depth here, and would highly appreciate any advice, insights, thoughts or warnings you can provide.
Thank you for reading though this wall of text:)
Matej
Hi.
It sounds like you are trying to design a lentiviral construct for inducible expression of a long noncoding RNA (lncRNA) linked to the expression of a fluorescent protein, in a doxycycline-dependent manner. One strategy you mentioned is to use two separate inducible promoters, one driving the expression of the lncRNA and the other driving the expression of the fluorescent protein. While this approach may be cleaner, it is true that using two separate TRE promoters may increase the risk of recombination between the promoters. However, using a recA mutant strain for cloning, such as Stbl3, can reduce the risk of recombination. Additionally, using a vector with an amp/kan selection system can help to prevent recombinant clones from propagating.
Another strategy you mentioned is to use a transcriptional polycistron, with a single TRE promoter driving the expression of both the lncRNA and the fluorescent protein. In this approach, a tRNA sequence is included between the lncRNA and the fluorescent protein to facilitate cleavage of the transcript and separation of the lncRNA and fluorescent protein products. One concern with this approach is that the fluorescent protein product may be missing the 5' cap or polyA tail, which could affect its stability and translation. One solution to this issue could be to include an internal ribosome entry site (IRES) sequence between the tRNA and the fluorescent protein, which would allow the fluorescent.
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