Method for establishing colorectal cancer HK2 reporter gene cell line
US11492597B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Nov 11, 2019 |
| Grant date | Nov 8, 2022 |
| Priority date | — |
| Expiry date | Sep 12, 2040 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N2510/00
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present invention discloses a method for establishing a colorectal cancer HK2 reporter gene cell line, specifically including: firstly, designing a site-specific sgRNA sequence of an HK2 gene, and cloning same into a PX459 plasmid; integrating a homologous recombination sequence of an HK2 gene and a green fluorescent protein DNA fragment (EGFP), and transforming the plasmid and the integrated fragment together into a colorectal cancer cell line HCT116 by electroporation; and performing signal cell screening through a flow cytometer to obtain EGFP-expressing cells, and amplifying a monoclonal cell line; and identifying a positive HK2 reporter gene cell line through PCR identification and Western blot, among screened EGFP-expressing cell lines. The colorectal cancer cell line HK2 gene and EGFP are co-expressed, and the expression level of the EGFP is highly consistent with that of the HK2 gene. Therefore, the expression level of the HK2 gene can be accurately determined by detecting a change in the expression level of the EGFP. The method for establishing a cell line in the present invention are simple, easy to implement and efficient, and a gene site can be precisely positioned.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.