What going on during the fregment integration steps after a plasmid transfection?

20 December 2023 2 9K Report

I would like to ask a question about constructing stable cell lines.

If someone has the whole genome sequencing results of their overexpression stable cell line that would be really helpful. That would give us a clear solid example of what going on during the fregment integration steps.

I would like to ask, when using vector transfection to construct stable cell lines, is gene recombination inclined to randomly insert the entire part of the transfected vector into one random position (I mean a whole block integrated into the genome, the target gene and the resistance gene will be integrated near by as they were in plasmid). Or is it inclined to random integration, that is, the target fragment and the resistance gene are integrated in different spots? In addition, in the final cell line obtained, how many copies of the fragment are integrated into the genome? (Because the results we often get like, final seed 30 clones that can survive under puromycin, but only 3-5 contain the target fragment, which seems to answer this question, that is, random fragments of random integration are high probability events in this case.)

Online information has reported that, due to the LTR sequences on the lentiviral vector, during gene recombination, the whole sequence between LTR can be integrated into a specific sequence position of the genome, could someone help me to double confirm this information?

About the role of resistance genes, could we understand it in this way? 1). In the overexpression period (2-7 days after transfection), screen out the clones that have not been successfully transfected; 2). In the integrated period (2-14 days after transfection), screen out the clones that have not successfully integrated the resistance gene. 3). After the stable cell line is constructed, maintain the purity of the single-cell clone. Am I right? I suppose that if random fragments of random integration theory is right. Then I would not expect that all the puromycin-resistant cells all have my target gene overexpressed.

I saw a product sells on the takara website which are linearized resistance markers used for co-transfection with other overexpression vectors. I thought that linear resistance markers would increase gene integration efficiency, then it may indirectly increase the probability of simultaneously integrating the interested fragment and the resistance gene in the same cell(may in different spots of genomic). Compared to those methods that transfect vectors contains resistance gene, above linearized resistance markers co-transfection methods would have more 'positive' cell clones to survive(have both interested gene and resistant marker) , then increasing the possibility of getting those clones.

In those easy transfect cells, such as HEK293 or CHO-K1, the transfection efficiency can easily reach more than 95%, so can we understand it in this way? Compared to co-transfecting overexpression plasmid along with a linearized resistance marker, to a vector containing both resistance marker and target fragments, for a single cell, the possibility to get both interested gene and resistant marker is almost the same, right? 95%*95%=90.25%

Thank you for your reading, and please let me know if I did not make my idea clear. It would be really helpful if you answered my question.

Best,

Le Chang

https://www.researchgate.net/post/Is-it-necessary-to-linearize-a-plasmid-before-making-stable-transfectants

https://www.thermofisher.cn/cn/zh/home/references/gibco-cell-culture-basics/transfection-basics/applications/plasmid-transfection.html

above two links may already answer my questions. But, I am appreciated if someone have more information want to share.

Robert Adolf Brinzer

1. Depends on what is in the plasmid you are using. If your plasmid is using some transposons then you can get almost random integration. The copy number will depend on if it is a jumping or replicating transposon. Viral vector derived plasmids tend to integrate into specific regions (eg. Lentivirus derived ones) but can be multiple copy. If integrated using recombinases (like the CRE/Lox system) then insertion will be site specific and usually single copy depending on the number of landing sites and if the genome is diploid. Similarly plasmids that integrate by homologous recombination will have 1-2 copies. The part of the plasmid integrated is usually a specific cassette flanked by regions used for integration (eg. homology arms, recombination sites or ITRs). Most plasmids try to avoid backbone integration into the host genome as that is prone to gene silencing by methylation.

2. You are correct everything between the LTRs is integrated. You will find most plasmids of that variety have a cassette with your GOI and the selection marker that is flanked by the LTRs with the replication machinery on a separate helper plasmid.

3. Your GOI and selection marker should be integrated together. After transfection treat each clone as a separate cell line and maintain selection for a least a month. It is good to periodically screen for the marker as that will eliminate any clones that have had the integrated cassette silenced.

4. I would have to look the maps of those specific vectors.

5. Yes the probabilities are combinatorial to an extent though with enough different construct being co-transfected there would eventually be signs of toxicity.

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