A kind of siRNA that specifically inhibits the expression of echs1 gene and its application
A gene expression and gene technology, applied in the field of siRNA, can solve the problems of high price, slow molding speed, poor model stability, etc., and achieve the effect of promoting growth and increasing lipid droplets
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Embodiment 1
[0030] Embodiment 1, siRNA design and synthesis
[0031] 1. Synthesis of ECHS1 siRNA
[0032]The full-length sequence of mouse ECHS1 mRNA (NM 053119.2; sequence 3 in the sequence table) was obtained from the database of the National Center for Biotechnology Information (NCBI) in the United States. According to the principle of RNAi, combined with design software, on the basis of experiments, the design, synthesis and An effective 21nt siRNA targeting mouse ECSH1 gene was screened out. A BLAST comparison check was performed to ensure that there was no homology to other genes.
[0033] The sequence of ECHS1 siRNA is: 5'-GCCUUUGAGAUGACGUUAAtt-3' (sense strand) (sequence 1);
[0034] 3'-gtCGGAAACUCUACUGCAAUU-5' (antisense strand) (SEQ ID NO: 2).
[0035] Chemical synthesis was carried out by Shanghai Gemma Pharmaceutical Technology Co., Ltd. (factory address: 602, No. 1011 Halei Road, Zhangjiang, Pudong, Zip Code: 201203): using NTP as raw material, using ABI...
Embodiment 2
[0040] Example 2, Effect of ECHS1 siRNA on the expression of ECHS1 gene in AML-12 cells in vitro
[0041] 1. Group transfection
[0042] Spread AML-12 cells evenly in a 24-well plate and divide them into 4 groups:
[0043] High-fat experimental group: containing 10% (volume percentage) inactivated newborn bovine serum, 100U / ml penicillin and 100mg / ml streptomycin, sodium oleate of 666 μ mol / L and sodium palmitate of 333 μ mol / L DMEM / F12 medium; transfect 200 pmol of ECHS1 siRNA of Example 1.
[0044] High-fat control group: containing 10% (volume percentage) inactivated newborn bovine serum, 100U / ml penicillin and 100mg / ml streptomycin, sodium oleate of 666 μ mol / L and the sodium palmitate of 333 μ mol / L DMEM / F12 medium; 200 pmol negative control siRNA of Example 1 was transfected.
[0045] Normal experimental group: DMEM / F12 medium containing 10% (volume percent) inactivated neonatal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin; transfected with 200 pmol of...
Embodiment 3
[0051] Example 3, Effect of ECHS1 siRNA on AML-12 Cell Steatosis in Vitro
[0052] 1. Group transfection
[0053] AML-12 cells were spread evenly in 12-well plates and divided into 4 groups. Concrete grouping is with the step one of embodiment 2.
[0054] 2. The effect of ECHS1 siRNA on lipid change of AML-12 cells in vitro
[0055] The cells after step 1 were subjected to the following experiments 1 or 2 or 3, respectively.
[0056] 1. Oil red dyeing
[0057] Add 4% paraformaldehyde to a 12-well plate and fix at 4°C for 30 min, wash twice with distilled water and then add oil red O dilution (oil red O 0.5g dissolved in isopropanol 100ml saturated solution and distilled water by 3: 2 diluted, left to stand for 5-10min and filtered for use), dark stained for 10-15min, washed once with 60% ethanol and differentiated under the microscope until the interstitium was clear, washed twice with Mayer’s hematoxylin counterstained nuclei for 8 seconds, washed twice Afterwards, inver...
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