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Vol 34(2018) N 2 p. 9-17; DOI 10.21519/0234-2758-2018-34-2-9-17
I.G. Vorobyova1*, R.R. Shukurov2, D.G. Kozlov3, Т.В. Koryagina1, N.V. Antipova1, and V.N. Stepanenko1

Modification of a System Based on the Use of Markers of Selection and Sorting for Screening of Stable Transfectants

1The Shemyakin-and- Ovchinnikov Institute of Bioorganic Chemistry, Russ. Acad. Sci., 117997, Moscow Russia
2The International Biotechnology Center "Generium" (LLCIBC Generium), 127006, Moscow Russia
3The State Research Institute for Genetics and Selection of Industrial Microorganisms of National Research Center "Kurchatov Institute ", 117545, Moscow Russia

*iva1647@yandex.ru
Received - 04.12.2017; Accepted - 01.03.2018

References

A p-hook technique based on the expression of protein domains on the cell surface has been modified and used for the selection and effective high-sensitive sorting of stably transfected CHO-S cells. A model EYFP protein served as a target produced protein and also as an expression-marking protein. Chimerical proteins TiBP-PDGFR and CBD-PDGFR that provided a capacity of binding to carriers, titanium and cellulosic support, were constructed. As a result of the combined selection with the use of the chimerical CBD-PDGFR protein that is exposed on the membrane a series of clonal lines with high and stable production of the model protein was obtained. The level of the model protein gene expression in three clones from the screened series exceeded by more than 30 times the mean level for the original cell pool. The stability of the expression was retained in the culture at the late (20th and 35th) passages. The use of the constructed chimerical CBD-PDGFR marker of selection and sorting permits to rapidly screen high-productive and stable transfectants, which reduces the cost of creating a collection of efficient cell lines.

adhesion, CHO cells, selection, sorting, titanium-binding domain, cellulose-binding domain, expression, exposed chimerical proteins.



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