Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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How would you overexpress and knock down a gene of interest. I know you have to use a plasmid vector that contain a promoter but I am not sure about the procedure. Please help me!
To overexpress a gene of interest, maybe u can clone ur gene into Baculovirus. Baculovirus has a strong promoter which will actively transcribe the gene of interest. Maybe you can find the protocol of using Baculovirus.
To knock down a gene, many different methods can be used also. But first how is your condition? Knock down a gene from?
You can use plasmid-based approaches for either overexpression or knockdown. You can also use oligo-based approaches for either (oligos are short linear strands of nucleic acids or nucleic acid analogues).
To overexpress, clone the cDNA of interest into an expression vector which will express its insert in the cell type you wish to use.
For knockdown, you can either express an antisense transcript or an shRNA, but these knockdowns will not be very specific. The antisense transcript will trigger RNase-H activity and the shRNA will use argonaute activity; both of these enzymatic systems will recognize mRNA with as little as 8 bases of complementarity.
To overexpress, you can deliver mRNA into cells or an organism. This can be tricky as the mRNA can be degraded, but electroporation or lipofection of cell lines or microinjection into early zygotes can give useful overexpression. There are no good methods yet for organism-wide overexpression in adult animals.
For knockdown, you can deliver antisense oligos into cells or use molecules that trigger the RNAi pathway such as siRNA or shRNA. The most specific approach is to deliver steric-blocking antisense oligos, which do not use enzymatic systems like argonaute or RNAse-H. Steric-blocking oligos have longer complementarity requirements for knockdowns. Steric-blocking oligos have modified backbones which do not trigger RNase-H activity. These oligos include 2'-O-methyl RNA, locked nucleic acids (LNA), peptide nucleic acids (PNA) and Morpholino oligos. Different steric-blocking oligo types have different optimal delivery techniques, though any of them can be delivered into cell culture by electroporation. In-vivo delivery moieties have been developed to bring steric-blocking oligos into cells after systemic injection (e.g. i.v., i.p.) into adult animals.
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