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Arabameri H, Pahlavanneshan S, Tavallaei M, Basiri M. Applications of the CRISPR-Cas9 System in Generation of Model Cell Lines for Cancer Biology. Multidiscip Cancer Investig. 2017; 1
URL: http://mcijournal.com/article-1-107-en.html
Abstract:   (848 Views)
Introduction: Cancer is known to be the second leading cause of death in the world after
the cardiovascular disease. Therefore, it is important to study the various factors involved
in cancer. In this regard, animal and cell models can be used to examine the different stages
of cancer, as well as its prevention and treatment. In the present study, we tried to stablish a
genome editing pipeline to generate genome edited cancer cell lines using CRISPR CAS9
technology.
Materials and Methods: We seeded about 40,000 cells in a 4-well plate and transfected
CRISPR CAS9 constructs by PEI to A549 cells on the next day followed by a brief selection
with puromycin. We allowed the remaining cells to multiply and form colonies then
we separated and transferred them in new plates using ring cloning method. On the obtained
colonies, we examined the function of the CRISPR, precisely designed the primer
for the CRISPR target area and sequenced the PCR product.
Results: In our study, we were able to colonize CRISPR-modified single cells and propagate
them into permanent cell lines harboring the modified CRISPR target. Using transient
expression of the antibiotic resistance gene, we successfully enriched the colonies with genome
modified clones to obtain a higher efficiency.
Conclusions: Proposed method provides a convenient means to generate different genome-
edited cancer cell lines which can be used to study the function of any candidate
gene in various stages of cancer, including metastasis, which plays a major role in increasing
mortality in cancer patients.
Full-Text [PDF 62 kb]   (266 Downloads)    
Type of Study: Original/Research Article | Subject: genetics
Received: 2017/10/28 | Accepted: 2017/10/28 | ePublished: 2017/10/28

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