Out of curiosity, I got this question whether knocking out (deletion) of a gene on one side and knocking down (RNAi) of the same gene on the other side will affect the cell in a similar manner or not.
If not why?
If yes, is it applied to every gene?
Knock out of a gene will correspond to no expression (gene deletion) in the cell, which can be appropriate to confirm the functional specificity, to observe processes which are affected in its absence and some other processes which are compensating the absence of the gene.
Knockdown of a gene will result in expression defect by decreasing the level of the transcript (mRNA silencing).
Now, the points which I am wondering upon are:
KO(deletion) results in completely taking out the effect of the gene of interest (GOI). Meaning, when you remove this gene there is no protein product of the gene. On the other hand, KD (RNAi), leads to reduction of effect of a GOI's expression but does not completely remove the effect of the gene. For example, gene "X" is useful for cell proliferation and survival. Upon KO of "X" all cell die due to lack of cell proliferation. But KD of "X" does not kill the cells but reduce its proliferation rate.
@Debottam Sinha
Thank you for replying to my question.
Following your example, if KO of 'X' is not viable but knockdown is,
why is there no survival chance in knockout or more elaborately, why knockouts cannot approach certain other mechanisms to survive (even slow proliferation) ? or
How the presence of silenced mRNA allow cells to proliferate by certain mechanism but its absence doesn't?
It will depend on the gene. Many genes contain non-coding RNA within their intron like miRNAs, snoRNAs...These non-coding RNAs are maturated from the intronic sequence during/after splicing. In the case of KO, they will be lost, in the case of KD they will still be expressed, so yes KO and KD will create different environments in this context.
After, when it comes to protein, KO and KD should lead to the same result in theory but not in practice since it is rarely possible to get a full KD of a given gene (always some leftover).
This all depends on several factors - for example - is the GOI crucial for cell proliferation? (there are about 400-500 genes currently considered as truly essential), are there homologues of the gene?
Also, the gene may be required only upon some conditions, e.g. presence of some metabolite (think of lactose in bacteria), or upon some stress etc.
If there is at least one additional homologue, the cell can simply upregulate it to compensate for the loss of GOI. If there are none however, the cell may not have any other way. For metabolism, there may be an alternative pathway.
The researcher can not create a perfect experimental setup without any genetic( or environmental) perturbations. I mean, the model organism will determine the method. If I want to examine an effect of a gene/genes in Saccharomyces c., I use gene KO(because of the higher rate of homologous recombination), and I am counting with the compensatory effects. Otherwise I will use gene silencing using C. elegans because it's easier to carry out.
Some genes can also compensate with increased expression in the presence of KD, but would obviously be unable to do so under KO. So, for a situation where KO is lethal, there is no functional duplicate that can fill the missing role, but KD can result in expression changes that prevent lethality, even if they lead to a phenotype/phenocopy. I have seen some researchers advocate for using both KO and KD to study a gene, when possible, because, in cases where the two mechanisms provide different answers, neither may be sufficient, on its own, to demonstrate the role and function of the gene in question.
according to the role of gene in the cells but generally both term will affect cell function depending of the type of protein produced by gene
If both methods work well, both should lead to comparable effects (even a knockout means the complete loss of function, whereas, a knockdown should lead to a reduced effect).
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