Serial dilutions: A simple and accurate method to achieve genetically homogenous clones from a cell population

سال انتشار: 1399
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 196

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شناسه ملی سند علمی:

ICIBS01_082

تاریخ نمایه سازی: 2 آذر 1399

چکیده مقاله:

Objective & introduction: Screening, diagnosis and treatment of genetic diseases have been facilitated by the emergence of gene editing technologies. Although these technologies can introduce mutations in desired locus, but evaluation of polyclonal cells with different indels and off- targets created by these mutations is a major challenge. Therefore, isolation of single edited cells may be necessary for evaluation of specific genotype and ultimately desired phenotype characterization. Multiple approaches have arisen for isolation of individual cells, such as fluorescent- activated cell sorting, serial dilutions, and cloning cylinders. Factors like simplicity, more economical, and the nature of transfected cells (suspension or adherent) are involved in choosing methods for cells isolation. Despite the widespread usage of these methods, there are some limitations, such as costly, requiring a fluorescent reporter for using FACS, and aseptic techniques for cloning cylinders methods. Similarly, serial dilution method is time-consuming and arduous, however, it is preferably used to separate single clone because it merely needs a statistical estimation and pipetting tools. In this study, we investigated a proper serial dilution method to isolate single cells from transfected HEK293 cells population by CRISPR/Cas9 technique.Material and methods: 1) 48-72 hours after transfection, we separated all cell lines in which have received desired vectors with selectable reporter like GFP or puromycin antibiotic. 2) It was prepared a concentration of 106 cells in 1ml growth media in a sterile falcon or 1.5ml microtubes. 3) Diluted cells 1:10, so we had 105, 1o4, 103 and 100 cells in falcon 15 respectively. In the last falcon, we had one cells for every 10μl, so we added 9 ml culture medium to this falcon up to the final volume of 10 ml. At the end of this step, we had one cell for every 100μl in every well of 96 plates.Results: After a week, examination of the cell plate under a microscope showed that approximately 23-30 wells contained single cells and after 14 days, this number would be increased to 40. Our results showed a good pipetting is imperative for better separation of cells.Conclusion: Due to heterogeneity variation in edited cells with genome editing tools, it is essential to examine the cells individually. Despite the limitation such as laborious and timewasting, researchers attribute to choose methods with features like ease of use, cheap, and lack of requirement for sophisticated tools like serial dilution.

نویسندگان

M Ranjbar

Genetics department, Shiraz University of Medical Sciences, Shiraz, Iran

F Amiri

Genetics department, Shiraz University of Medical Sciences, Shiraz, Iran

M Dianatpour

Genetics department, Shiraz University of Medical Sciences, Shiraz, Iran