The method of gene knockout is just like this.

Release date: 2017-09-19

The current genome editing tools alone do not change the arrangement of gene sequences. All they can do is create a few gaps in the genome sequence. The true change in the genomic sequence, the editing of the genomic sequence is the cell itself.

What we can do is to maximize the induction of cells to complete the genome editing as we wish.

So, what is the induction method?

Let's start with gene knockout~

If we are going to do a gene knockout.

The easiest but most laissez-faire way is to make an incision on the gene fragment we need to knock out and let the cells repair it on their own. In this process, the cell may have several bases deleted from the gap and several bases inserted. If the deleted or inserted base is not a multiple of 3, the gene may be missing due to a frameshift mutation, thereby achieving knockout. We randomly selected some of these treated cells for individual cell culture and expansion. These cell populations formed by the expansion of individual cells are referred to as a single clone. By analyzing the expression of these monoclonal genes by western, PCR, etc., we can find the cell populations in which the target genes are knocked out, that is, the knockout monoclonal cell lines that we need to construct. The function of the gene of interest in these monoclonal cell lines was deleted, but the sequence encoding the gene was still left in the genome, but it was destroyed and could not be expressed normally.

Another more controllable approach is that we create a separate incision at two distant sites in the gene sequence, and large segments between the two incisions are highly likely to be deleted. We can screen out these single clones with large fragment deletions. Their target genes can be completely deleted from the genome. However, this method requires more monoclonals to be validated because the probability of two sites being cut at the same time is lower than the probability that a single site is cut.

Both of the methods described above require random picking of the clones for verification. This is a bit of luck.

One way to make our monoclonal selection more purposeful is to label our edited cells.

For example, gfp fluorescent protein? Resistance genes and the like, and a homologous arm of the gene sequence of interest is added to both sides thereof to form a tag sequence. Putting them into the cell, they also put in some genome editing tools to create gaps in the sequence of interest, increase the probability of homologous recombination, and integrate these tags into the target gene sequence. Such large fragment insertion can simultaneously destroy the expression of the target gene, resulting in its lack of function. In this way, we can find our gene knockout cells through resistance screening or screening based on fluorescent expression.

Source: Boya Collection Technology (Micro Signal EdigeneTech)