I am interested to understand the genetic pathophysiology of Fuchs Dystrophy and for that purpose it is required a cell culture based model system of Fuchs corneal endothelium cell line. We have control corneal endothelium cell line but unfortunately still now we can not arrange a Fuchs cell line for experiment. Can I transform my control cell line into Fuchs cell line by any drug or other treatment? We do not have primary cell culture or immortalization facility.
Maynak, I am not aware of transformation of normal HCEnC line using chemical treatment. I also don't expect such an approach to reliably recapitulate the FECD phenotype. There are at least two labs (one in Japan and one in the US) that have established an FECD cell line from patients. You may consider requesting the lines from either one of these two labs.
Another option may be to edit the genome of your control cell line using CRISPR-Cas9 by introducing a mutation associated with FECD. As the TCF4 trinucleotide repeat extension being the most prevalent in FECD you may consider attempting to introduce this into your control cell line, but I suspect it to be the most difficult to introduce by HDR. Other genes have been associated with FECD and the mutations identified in those genes would likely be much easier to introduce using HDR, but those mutations are not as prevalent in FECD as the TCF4 trinucleotide repeat.
Yet another option is to use primary cells. It is suspected that FECD is a result of premature cell senescence, as primary cells undergo cell senescence, you may consider late passage primary cells to sufficiently recapitulate some of the features of FECD, while early passage cells would be your controls. However, this approach has not been vetted, so a fair amount of characterization would be needed.
Best.
Thank you Ricardo.Actually I have already requested two labs for the cell lines but no one gives me a positive response.Primary endothelium culture is difficult one but it is the only remaining option for me.
That is surprising. You may need to consider in greater detail what you can do to facilitate transfer of the cell lines. For example, most institutions have so-called materials transfer agreements (MTAs), for which you or your institution will have to provide information. Assuming you have done this or are ready and willing to provide all information as requested but have either received no response or your request has been rejected, then another option is to contact the journal in which the cell lines were first published. Most reputable journals have a stipulation that unless otherwise stated (and justified) all reagents published in their journal should be made available to other scientists requesting them. For example, below is a copy of the statement regarding reagent sharing for work published in Nature journals:
"An inherent principle of publication is that others should be able to replicate and build upon the authors' published claims. A condition of publication in a Nature journal is that authors are required to make materials, data, code, and associated protocols promptly available to readers without undue qualifications. Any restrictions on the availability of materials or information must be disclosed to the editors at the time of submission. Any restrictions must also be disclosed in the submitted manuscript.
After publication, readers who encounter refusal by the authors to comply with these policies should contact the chief editor of the journal. In cases where editors are unable to resolve a complaint, the journal may refer the matter to the authors' funding institution and/or publish a formal statement of correction, attached online to the publication, stating that readers have been unable to obtain necessary materials to replicate the findings."
Hope that you are successful in obtaining the cell lines so that you may proceed with your work.
Best.
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