Genetic updating and marks of cellular lines

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Abstract

Abstract. Despite great advances in the biology of stem cells, there are still many dark spots. Genetic modification techniques, which can be used to track the lines of different cells, primarily stem cells, help to solve this problem. Various methods of biotechnology research are considered, allowing to evaluate the options of introducing new genes into cells and even whole organisms, as well as methods of controlling their expression in time and space, their activation, differentiation and decrease in functional activity, expression of several target genes. Options with multi-cystron vectors encoding several proteins are described. Options for introducing genes using plasmids, electroportation of their disadvantages and advantages are characterized. The most promising and the safest is a retroviral vector using lentivirus vectors capable of generating additional copies of itself, which is very important in the field of biotechnology security. A line of packing cells, usually 293T cells, is used to produce a viral vector. Prospects for the use of adenovirus and adenoassociated vectors are characterized. The achievement of modern biotechnology methods is the system of short palindrome repetitions located in groups, which is a unique tool for genome editing. At the heart of this system is the process of cutting out sequences of deoxyribonucleic acid, which are permanent and which are supported by cells regardless of subsequent divisions or changes in condition. The system allows geneticists and medical researchers to edit parts of the genome by removing, adding or modifying successive sites of deoxyribonucleic acid. An important problem with biotechnology methods is how to control the expression of transgenes. Today, it is quite effective to control expression with a factor present in the gene delivery vector itself and which is only active in a certain type of cell. Endonuclease bacteriophage P1 is used to regulate transgene expression, which cuts deoxyribonucleic acid only at specific sites. This system is introduced in both eukaryotic and prokaryotic systems.

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About the authors

A. V. Moskalev

Military medical academy of S.M. Kirov

Author for correspondence.
Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

B. Yu. Gumilevskiy

Military medical academy of S.M. Kirov

Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

A. V. Apchel

Northwest Medical Training Center for Postgraduate Education

Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

V. N. Tsygan

Military medical academy of S.M. Kirov

Email: vmeda-nio@mil.ru
Russian Federation, Saint Petersburg

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Supplementary files

Supplementary Files
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1. ig. 1. Scheme of the structure of bicistronic vectors on VSPR and 2A-sequences: a - expression of mammalian plasmids; b - internal site of ribosome landing and 2A-sequence; c - retrovirus genome. LTR - long terminal repeat In the vector, the genes gag (capsid protein), pol (reverse transcriptase) and env coat protein) are replaced by the gene of interest; r - vector of lentiviruses. RRE - Rep response element, cPPT - polypurine region, CMV - promoter for gene of interest, WPRE - enhancer sequence; e - ITR - inverted terminal repeat (inverted terminal repeats), E1-4 are early expressed genes (early gene expression, L1-5 are late genes). ; e - AAV genome ITR repeat Two genes of the vector are replaced by the gene of "interest"

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2. Fig. 2. Transcription options: a - P - promoter; TA - tetracycline activator; DOX, doxycycline; TRE - reaction element Tet; GOI - gene of "interest"; b - TSP - tissue promoter; UP - ubiquitous promoter; c - TAM - tamoxifen; d - RISC RNA-induced inhibitory complex; AGO2 - Argonaute 2 (endonuclease, protein complex); e - CRISPR-Cas9. PAM - adjacent protoscanner motif, sgRNA - single-stranded RNA, DSB - double-stranded break; Non-homologous end joining (NHEJ) - non-homologous end joining; homologous recombination (HR) - homologous recombination

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Copyright (c) 2020 Moskalev A.V., Gumilevskiy B.Y., Apchel A.V., Tsygan V.N.

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