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G6PD-deficient Caki-1 stable cell strain and construction method thereof

A technology of caki-1, cell line, applied in the field of medical molecular biology

Inactive Publication Date: 2015-04-29
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, is the expression and activity of G6PD abnormal in the corresponding cell lines of renal cell carcinoma, what is the specific role of G6PD in the development of renal cell carcinoma, and what is its regulatory mechanism? The above questions have not been reported at home and abroad so far

Method used

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  • G6PD-deficient Caki-1 stable cell strain and construction method thereof
  • G6PD-deficient Caki-1 stable cell strain and construction method thereof
  • G6PD-deficient Caki-1 stable cell strain and construction method thereof

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Embodiment Construction

[0030] 1. Design and synthesis of siRNA sequence and DNA template

[0031] OligoEngine RNAi software was used to design the siRNA interference sequence 5'-GCCTCAGTGCCACTTGACA-3' for the 3' non-coding region of the human G6PD gene, and the irrelevant sequence 5'-TTCTCCGAACGTGTCACGT-3' was used as a control, and then two complementary and containing The DNA templates of sense strand and antisense strand of interfering sequence or irrelevant sequence are connected by a hairpin structure of 9 deoxynucleotides in the middle, followed by RNAPoly III transcription stop point, and Bgl I and Hind III enzyme cutting sites are introduced at both ends point; the DNA template corresponding to the interfering sequence in the 3' non-coding region of the human G6PD gene is the interfering F chain 5'-GATCCCCGCCTCAGTGCCACTTGACATTCAAGAGATGTCAAGTGGCACTGAGGCTTTTTTA-3' and the interfering R chain 5'-AGCTTAAAAAAGCCTCAGTGCCACTTGACATCTCTTGAATGTCAAGTGGCACTGAGGCGGG-3'; and the DNA template corresponding ...

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Abstract

The invention relates to the field of medical molecular biology and particularly relates to construction of cell research model by using a siRNA interference technology. The G6PD-deficient Caki-1 stable cell strain is a stable cell strain which contains DNA double stranded fragments and green fluorescent protein (GFP) gene expressed by target interfere G6PD gene and can target and silence Caki-1 cell strain so that expression of endogenous G6PD is not less than 80% by virtue of the siRNA interference technology, wherein the DNA double stranded fragments comprise the interference F chain 5'-GATCCCCGCCTCAGTGCCACTTGACATTCAAGAGATGTCAAGTGGCACTGAGGCTTTTTTA-3' and interference R chain 5'-AGCTTAAAAAAGCCTCAGTGCCACTTGACATCTCTTGAATGTCAAGTGGCACTGAGGCGGG-3'. By the siRNA interference technology, the expression of silencing human renal clear cell carcinoma Caki-1 cell endogenous G6PD is not less than 80%, the G6PD-deficient Caki-1 stable cell strain (Caki-1-G6PD siRAN) is constructed. The cell model can be used to study the correlation between G6PD and renal cell carcinoma, as well as the specific effect of G6PD in incidence and development of renal cell carcinoma and related regulatory mechanism.

Description

technical field [0001] The invention relates to related technologies of medical molecular biology, in particular to constructing cell research models by using siRNA interference technology. Background technique [0002] Glucose-6-phosphate dehydrogenase (G6PD) is a key rate-limiting enzyme of the pentose-phosphate pathway (PPP), located in the gene high-density region Xq2.8 (NM_000402), The full length is 18kb, consisting of 13 exons and 12 introns. G6PD is a housekeeping gene, which is expressed in all tissue cells. The full length of its mRNA is 2395bp, and its cDNA is 1548bp, encoding 515 amino acids. The pentose phosphate pathway can produce ribose pentaphosphate and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which are important raw materials for nucleic acid synthesis and reducing agents for cell growth and survival. Tumor cells are cells with uncontrolled proliferation and differentiation, and there is an imbalance of redox stress. In recent years,...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor 朱月春况应敏张巧王艳玲杨丽娟陈龙韩巧巧
Owner KUNMING MEDICAL UNIVERSITY