Simply Stable

(August 22nd, 2017) Making stable cell lines is a laborious task. Canadian researchers came up with a new method using CRISPR/Cas9-mediated knock-in.





Anyone who has tried working with stably-transfected cell lines knows they are not quite the easy option they promised to be. Sure, it sounds appealing - just thaw the cells, grow them up, and off you go. No fussy dissections, lots of material to experiment on, every batch the same.

Only it isn't. Stable cell lines may be more convenient than real tissues, but they have a few disadvantages. For one thing, they are not homogeneous. When you make a stable cell line, you randomly insert a construct into the genome, and because it is random it can end up anywhere and in different numbers. One cell might produce loads of your protein, the one next to it is little different from the wild type.

Even just making that stable cell line in the first place is a lot of work, too. So it comes as a breath of fresh air to hear that a new way of making stable cell lines has been announced by Brian Edwin Chen's group at McGill University, Canada. And at the heart of this new method is - yes, well, you've guessed it, CRISPR.

The idea behind Chen's exciting new way of making stably-transfected cell lines is actually really simple. You use CRISPR to insert the construct just where you want it in the genome, under the control of a specific promoter.

How does it do that? Pretty much as you would expect - you create a CRISPR construct to cut the genome at a predetermined location, then let homologous repair to insert the construct. The construct includes three important elements. First, a reporter gene such as a fluorescent protein. This will tell you how much of a product the cell is making. Second, there is the payload itself. Finally, there is an antibiotic resistance gene. Once transfection has been achieved, you apply the usual antibiotic selection, but this time with a difference: once selected, you take the antibiotic away. For good. The reason you can do this with this method and not with traditional transformation is because in the latter case, cells usually have methods of ditching genes that interfere with the genomic structure. With this new method, you place the gene carefully into the genome so that it hitches a ride with a housekeeping gene.

To illustrate the point, Chen made a construct consisting of a red fluorescent protein, a protein quantitation reporter incorporated between the endogenous and foreign genes, and a Zeocin resistance gene. They used CRISPR to insert the construct into two genes: the human ribosomal protein L13A and ? actin, two well-characterised housekeeping genes. They first used conventional PCR to check that the construct had been inserted at the right location. They selected for transfected cells, then took away the antibiotic, and over 30 days afterwards the cells were glowing a nice, healthy red.

They did a full dress rehearsal with green fluorescent protein as the payload, and confirmed that cells glowed red in the nucleus and green in the cytoplasm.

Importantly, the levels of red and green were closely correlated. Why is this important? The answer to that question brings out an attractive feature of this method. Because the construct has one copy of the reporter (RFP) and one of the payload (GFP), the level of reporter fluorescence is a faithful indicator of the level of expression of the payload.

There are a number of advantages to this method. First, using CRISPR to target the transgene to a specific site reduces the notorious heterogeneity of the resultant cell line. Exactly one gene is inserted in all cells, and always under the control of the same promoter. Second, once you have performed your antibiotic selection, you can take the antibiotic away - no risk of developing new loci of resistance or of killing off your cells with antibiotic poisoning. Third, the protein quantifier construct means you have a good idea how much transgene is being expressed.


Steven D Buckingham

Photo: National Institutes of Health




Last Changes: 09.11.2017



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