METHOD OF DETERMINING LEVEL OF CHIMERIC GENE Trim5a EXPRESSION Russian patent published in 2016 - IPC C12Q1/68 

FIELD: medicine.

SUBSTANCE: invention refers to medical and molecular genetics, namely to genetic structures. Method of determining level of expression of genes Trim5a-ch and Trim5a-hum in samples of the cell suspension previously treated with gene therapy drug, which includes a chimeric gene Trim5a, provides: extraction of nucleic acids from a cell suspension samples; treatment of recovered nucleic acids using DNAse to produce total cell RNA without DNA impurities; carrying out a polymerase chain reaction, combined with reverse transcription, using the obtained total cell RNA of products in real time to produce curves of accumulation of fluorescent signal; calculation of normalized concentrations of mRNA TRIM5a-ch and mRNA TRIM5a-hum in test samples, characterising the expression level, based on values of cDNA TRIM5a-ch and cDNA TRIM5a-hum and cDNA of beta-gene glucuronidase in PCR sample made from accumulation curves of the fluorescent signal ; calculation is made by formula: normalized TRIM5a mRNA concentration = number of copies of cDNA TRIM5a/number of copies of cDNA gus, where gus, is "house-keeping" gene of human beta-glucuronidase.

EFFECT: this method enables to determine the number of mRNAs of chimeric gene TRIM5a and number of mRNAs of human gene TRIM5a; to compare expression level in different conditions and specifying expression conditions so that transcription is sufficient for developing antiviral activity of gene within the range of physiological values safe for cells.

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