FIELD: biotechnology.
SUBSTANCE: invention relates to biotechnology, in particular to a method for creating a cell model of leukaemia for modelling and studying processes occurring in leukaemia cells. Method is aimed at detecting those single cells in the population in which translocation has occurred, and their selection. Primary selection is carried out by fluorescence of the reporter protein, and ensuring that all elements (Cas9 and RNA guide genes) required for translocation induction have entered the cell is ensured by the fact that all these elements together with the fluorescent protein gene are assembled into one plasmid. Such a plasmid for inducing a new translocation is created on the basis of a universal vector in two cloning steps. To detect translocation, PCR is used, and without a separate step of DNA extraction (so-called direct PCR), combining this approach with embedded PCR, which increases sensitivity of the method. For reliable production of a cell line with a translocation with a probability of not less than 0.95, a preliminary check of the effectiveness of RNA guides and counting the frequency of translocations can be carried out. Based on the frequency of translocations, an optimal cloning strategy into subpopulations and a strategy of PCR analysis with combining several samples into one reaction are selected. Cloning is carried out using a cell sorter. Obtained monoclonal lines are characterized for the presence of translocation, the rearrangement region is sequenced, the expression of the fused gene product is shown and can be further used for the declared purposes.
EFFECT: invention enables to obtain monoclonal lines with translocations in the shortest possible time and with minimal time and laboratory resources.
21 cl, 5 dwg, 1 ex
