Breast cancer cell line with low expression of HK2 and siRNA used by same
A breast cancer cell, low-expression technology, applied in the field of medical molecular biology and genetic engineering, can solve the problems of lack of targets for breast cancer treatment, limiting the effectiveness and extensiveness of breast cancer treatment methods, etc.
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Embodiment 1
[0064] Example 1 Design and Synthesis of SiRNA (HK2 siRNA) targeted by HK2 gene mRNA
[0065] The inventors of the inventors of the inventors designed HK2 siRNA according to human HK2 sequence in GenBank, and the basic principles of design are as follows: (1) Starting from the AUG start password of the transcript of human HK2, looking for "AA" two-line sequence, It also records the 19 base sequences of the 3 'end, as a potential siRNA target site; (2) Do not design siRNA, do not target the non-encoding area of the 5' and 3 'ends, because these places have a rich regulatory protein binding area. And these UTR binding proteins or translation start complexes may affect the SiRNP nucleic acid ingenuclease complex binding mRNA to affect the effect of siRNA; (3) generally gc content in 10 nucleotides at 30% -70% In the range, a valid fragment is easier to find in a fragment of the GC content; (4) To avoid continuous repetition of the siRNA target sequence to form a secondary structure...
Embodiment 2
[0071] Example 2, 3 HK2 siRNA detection of HK2 interference effect in human breast cancer cells Zr-75-1
[0072] Human breast cancer cells Zr-75-1 is the product of the US ATCC cell library.
[0073] 1, transfection
[0074] (1) Treatment of human breast cancer cells Zr-75-1 in a culture dish (diameter of 6 cm) (6cm) containing 10% (v / v) neovasculum serum (diameter is 6 cm) (inoculated cell density in transfection) It is suitable for 70-80%.
[0075] (2) After completing step (1), SiRNA (NC siRNA, HK2 siRNA1, HK2 siRNA2 or HK2 siRNA3) was transfected with Lipofectamine 3000 (US Invitrogen) transfection (transfection method refer to Lipofectamine 3000 transfection reagent) Instructions), the cells were collected in 48 hours after transfection, and siRNA transfected human breast cancer cells Zr-75-1 were obtained.
[0076] 2, cDNA's acquisition
[0077] The total RNA transfected with Trizol (US INVITROGEN) was extracted from the total RNA transfected with human breast cancer cells...
Embodiment 3
[0104] Example 3, HK2 siRNA effect on Zr-75-1 proliferation of human breast cancer cells
[0105]The human breast cancer cells Zr-75-1 in step 1 obtained in Example 2 were inoculated to 96-well plates, and approximately 3,000 per well inoculated, and conventionally cultured to the adhesive wall; after the Cell County Kit-8 (Japan DOJINDO) Detected the OD value at 450 nm. After 5 days of continuous detection, the growth curve is drawn.
[0106] Growth curve figure 2 . The results showed that HK2 siRNA1, HK2 siRNA2 and HK2 SiRNA1, HK2 siRNA2 and HK2SiRNA3 can inhibit proliferation of human breast cancer cells Zr-75-1, and HK2 siRNA1 has the most obvious inhibitory effect on human breast cancer cells Zr-75-1 .
[0107] In combination of the above experimental results, HK2 siRNA1 was selected as a subsequent study of siRNA.
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