Hello, I am trying to knock-in a gene in the safe harbour region AAVS1 using CRISPR, to correct a loss-of-function mutation of my gene of interest. Could an already spliced mRNA sequence of my GOI in the form of cDNA be used as a transgene? Or is it better to insert a whole gene sequence? If yes do I still flank with promoter and polyA signals? Many thanks!
Dear Mr. Dario Pacitti,
Yes, it should be possible to use already spliced mRNA sequence as cDNA for transgene expression. But before using that mRNA sequence, to be absolutely sure, please compare the sequence with reference sequences at Ensembl BLAST or NCBI BLAST.
I think, you will have to insert the appropriate promoter region + cDNA sequence + poly A tail to get the expression of needed protein.
Best regards,
Kshitiz
Thank you all for the replies. I am aware of the need of a promoter and poly a tail for cDNA. However if you use a whole gene, would you still flank with promoter and polyA signals?
Usually whole gene sequences or mRNA sequences provided on databases have 5'UTR and and 3'UTR sequences, which usually should contain promoter and polyA tail, but it is better to explore it specifically for the gene sequence, you will choose.
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