Hi, Sanna.

Most cell lines maintained in in-vitro condition show varying degrees of aneuploidy. In particular, the cell lines contain virus or are cancerous chromosomal abnormality is prevalent. The causes are many-folds, recently entosis is added in the list. Check the review below. Even a specific cell stock you start with contain a similar degree of aneuploidy, becomes more heterogeneous cell population as culture continues. ATTC says its HEK293 cells contain adenovirus warning use of proper containment for handling. Any cell lines can be used for any biological study, even in genetic modification since you still have control group of cells or original your stock of cells to compare with those of experimental group. No need to worry too much unless you are critically determining gene copy or location in the chromosomes or genomic study in detail.

Thank you for your answer, Sang Ho Lee. I understand that most laboratory cell lines contain mutations and are prone to mutagenesis over time, but I would still like to know if HEK293 cells in particular have 4 copies of Chromosome 17 and 22.

Hi, Sanna.

Yes, there are!!!

In the original paper cited, their cell line may have dominant mutant mutated cell population of 4xC17 and 4xC22. However, that was the state of a particular cell population they studied. The particular cell stock they analyzed of HEK293 cell line might disappear now. Who knows until similar studies show. The supplementary figures show one of the peculiar chromosomal analysis by multiplex FISH as an evidence for the presence of the cells. It should not be interpreted as all the cells present in their HEK293 cell line shows tetrasomy in C17 and C22. As I answered it is prevalent cell lines maintained over long time. Unbelievable? Just read a part of description I cited below!!!

"Genetically, the MCF-7 line is not exactly the same as the intial isolated clone. Originally, it was described as having a karyotype containing 85 chromosomes which has since been reduced by 16 chromosomes. Today’s MCF-7 cell line has a karyotype containing 69 chromosomes. Furthermore, there are genetic discrepancies between the MCF-7 cell line from the Michigan Cancer Foundation and the ATCC cell line. This indicates that the ATCC cell line is of a different source than the other MCF-7 cell lines."

http://www.mcf7.com/

Hi Sanna, 

I have had a look at the supplementary information for the paper you referenced in your question (M-FISH data analysis - Supplementary spreadsheet). They commonly found an additional copy of both chromosomes 17 and 22 in HEK293 cells (so, 3 copies), although this was only the case in approximately half of the metaphase cells they assessed. In some HEK293 cells, there were only two copies of these chromosomes, whereas in others there was only a single copy. Therefore, as you can see it's a bit of a mixed picture but it seems that in their paper, the authors suggest three copies of the chromosomes you're interested in exist the majority of the time. I should say that this is for whole, intact chromosomes! 

In that paper, it also states that in half the metaphase cells they analysed, there was a translocation between chromosomes 11 and 22. Additionally, there was a translocation between chromosomes 11 and 17 in a quarter of cases. With this in mind, you could say that there are 3 whole copies of each of chromosomes 17 and 22, with an additional partial copy of each of them (due to translocations). So there aren't quite 4 whole copies. 

Of course, this was what they found in their cell line and it might not be the same as where you get your HEK293 cells from, or if there has been genetic drift during cell culture. If you are going to use HEK293 cells in some genetic experiments, the best thing to do would be to perform your own FISH upon them, to probe for your gene of interest and check how many copies of it exist in your stocks of this cell line. 

Please note that different chromosome copy numbers and translocations exist in the other cell lines derived from HEK293 cells (HEK293T & HEK293S). 

I hope this at least partially answers your question! 

All the best, 

Jonathan 

Sang Ho Lee and Jonathan Whitchurch, thank you both so much! I agree that I will need to do FISH on my own HEK293 cell line to determine how many chromosomal copies there are, as you mentioned Jonathan. I understand your point now, Sang Ho Lee; I see that these cell lines can change drastically in chromosome number between different lab's HEK293 cell lines and indeed, even after time in the same cell line. I am surprised that we as scientists rely on such abnormal cell lines when conducting research. Personally, I always considered HEK293 cell less abnormal compared to HeLa and HCT cells, however I now believe all immortalized laboratory cell lines have a lot of issues.

My lab is trying to knock out a gene found on Chromosome 22 in HEK293 cells using CRISPR-Cas9. I suspected that having extra copies of Chromosome 22 would make it more difficult to knockout completely.

It would be wonderful if your target gene is only one copy in the genome. However, gene manipulation by microinjection or transfection by using liposome, electrical shock, viral infection and so on always involves excess amount of nucleic acids and peptides in your case. Therefore, it is not a critical consideration that you may get incomplete knockout due to the presence of three copies. The recommended amount of  ingredients are enough to work in 3-fold within the cell. It will, then, be a completely novel achievement when you show complete KO in the HEK293 genome. This is why I am saying no worry too much. We are always using much excess of ingredients to achieve complete transfection whether it aims 'gain of function' or 'loss of function' in the genome.

Recently we have karyotyped HEK293 cells by conventional cytogenetics and arrayCGH. The majority of cells contain three copies of Ch17 and 22, and one addidional translocation of Ch17. Thus, there are 3 whole copies of each of Ch17 and 22, with an additional partial copy of Ch17.

In addition, the mean chromosome number and chromosome aberrations are significantly distinct in HEK293 cells from the different sources arguing that the long-term cultivated and propagated clones of HEK293 cells could undergo significant karyotype changes and selection.

Please see the following manuscripts for details:

Stepanenko A, Andreieva S, Korets K, Mykytenko D, Huleyuk N, Vassetzky Y, Kavsan V. Step-wise and punctuated genome evolution drive phenotype changes of tumor cells. Mutat Res. 2015 Jan;771:56-69. doi: 10.1016/j.mrfmmm.2014.12.006.

Stepanenko AA, Dmitrenko VV HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene. 2015 Sep 15;569(2):182-90. doi: 10.1016/j.gene.2015.05.065.

I am experiencing difficulty in using 293T cell for genome editing. I am asked to knock in a fluorescent tag to one target gene using 293T cells, and this gene is located in chromosome 22. after 4 months transfection of CRISPR-cas9 and donor plasmids, some cells still express the fluorescent protein. However, there are many in-confirmation futures during I identified whether the tag has been knocked in the gene.

I though that 293T cell is a cell line with abnormal genomes, it is not a good cell line as genome editing. Any comments?