I can happily knock out a lncRNA via transfection with siRNAs, but now need to do the mirror opposite.
Any pointers would be most welcome.
Thanks.
Dear Dr. Perry
Recently developed targeted genome-editing technologies using engineered nucleases, such as transcription activator-like effector nucleases (TALENs), provide a precise way to manipulate chromatin regions of interest. You should insert a CMV promoter and egfp just upstream of the lincRNA to achieve a single allele insertion to express the fusion egfp-linc RNA in cis. As it has been shown that the insertion of a double poly(A) site cassette into the genome by zinc finger nucleases (ZNF) led to an efficient transcriptional stop. You ought to insert such a double poly(A) site cassette downstream of egfp to terminate the transcription of lincRNA at the same genomic locus to obtain the control cell line. For further information please see Xiang et al, (2014) "Human colorectal cancer-specific CCAT1-LlncRNA regulates long-range chromatin interactions at the MYC locus". report as attached file.
Regards,
Hadi
Article Human colorectal cancer-specific CCAT1-L lncRNA regulates lo...
Hi Mark,
We have had success overexpressing lncRNAs in mammalian cells. We used mouse Neuro2a cells since we were expressing human lncRNAs, thus making detection by RT-PCR less complicated. We used Gibson assembly cloning to insert our lncRNAs into pCAGEN, which gives great expression in many mammalian cell types. I can share details of our approach by email if you would like.
Best,
Chris
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