High sensitive fusion cell screening method

Abstract

The invention discloses a high sensitive fusion cell screening method, belongs to the field of gene engineering, and relates to a fusion cell quick screening scheme based on Cre recombinase and Loxp sequence specific recognition. Through incision of the Cre recombinase and the Loxp sequence, fluorescence switching and cell resistance switching of the fusion cell are achieved, and therefore singlecell screening and subsequent expanding culture of the fusion cell are achieved. Universal expression Cre protein nude mice are used for injecting modified tumor cells into the mouse bodies so that tumor cell metastasis and development caused by cell fusion function can be observed. The invention provides a lentivirus plasmid vector, wherein the nucleotide sequence of a fluorescence imaging analysis carrier A which is capable of achieving fusion cell high sensitive rapid screening and in vivo tumor cell metastasis is shown as Seq ID No.1, and the the nucleotide sequence of a carrier B is shownas Seq ID No.2.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a scheme for rapid screening of fusion cells based on the specific recognition of Cre recombinase and Loxp sequences. In particular, it relates to a method of cleavage of Cre recombinase and Loxp sequence to realize fluorescence switching and cell resistance switching of fused cells, so as to realize single cell screening and subsequent expansion of fused cells. At the same time, through the use of nude mice expressing Cre protein, the transformed tumor cells are injected into the body, and the metastasis and development of tumor cells caused by cell fusion can be observed. Background technique [0002] 1. Cell fusion and tumor [0003] Cell fusion refers to the process in which two or more cells contact each other, and then the cell membrane, cytoplasm and nucleus fuse to form a hybrid cell, also known as cell hybridization. Although the occurrence probability of...

AI Technical Summary

Problems solved by technology

This method has obvious defects. First, both A cells and B cells have fluorescence. During the screening process of positive cells, the excitation light of two fluorescent proteins needs to be excited at the same time, which will cause strong background fluorescence interference. cells have more false positives

And due to the low efficiency of cell fusion, it is difficult to successfully screen a small amount of fusion cells

At the same time, due to the expression regulation and silencing effect in cells, if the traditional screening method is used, the time point of screening is high. Once the time is too long, the silencing effect in the cell cannot be ignored. If the fused cells cannot stabilize If two fluorescent proteins are expressed at the same time, the fused cells will be indistinguishable from the background cells, making it difficult to achieve the purpose of screening

At the same time, because the fused cells have the same antibiotic resistance as the original cells, it is impossible to screen a very small amount of fused cells by antibiotics, and can only be screened by flow cytometry. When the number of fused cells is very small, flow cytometry Cytometers are often difficult to detect

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