Interference fragment of colipase-like 2 (Clpsl2) gene and application of interference fragment
A lipid-like, gene-based technology, applied in gene therapy, DNA/RNA fragments, diseases, etc.
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Embodiment 1
[0020] Embodiment 1: In situ hybridization detects the expression localization of Clpsl2mRNA in epididymis tissue
[0021] Using ApaI to transfer pGEM-T-Clpsl2 vector containing Clpsl2 gene (Guan Yiqing, Yu Yanhong, Huang Dan, Pan Shanpei. Prokaryotic expression of mouse epididymis-specific colipase and preparation of its antibody[J]. Journal of Jinan University (Nature Science and Medicine Edition), 2010,31(2):105-110.) The plasmid was linearized, purified with phenol chloroform and labeled with Roche’s Digoxigenin Probe Kit, and the antisense probe was labeled with SP6 RNA polymerase to T7RNA Polymerase-labeled sense probe was used as a control, ethanol precipitated, and DEPC was dissolved in water.
[0022] The epididymis of 8-week-old sexually mature mice were taken, put into 4% paraformaldehyde by weight, fixed at 4°C for 2 hours, and washed 3 times with PBS, 5 minutes each time. Then add 30% sucrose solution by mass, and place at 4°C until the tissue pieces sink to the ...
Embodiment 2
[0023] Embodiment 2: Western blot detects the expression of Clpsl2 in each section of epididymis tissue
[0024]Eight-week-old sexually mature mice were killed by neck dislocation, and the epididymis were separated immediately. After removing excess fat and connective tissue, they were washed in pre-cooled PBS, and then the epididymis was separated into three parts: head, body and tail. Each sample taken was added to the Western and IP cell lysate (Mitsu) with a final concentration of 1mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor mixture (Calbiochem, Merck Millipore, USA) at a ratio of mass volume ratio of 1:10. organisms), grind in an ice bath. After centrifuging at 12,000rpm at 4°C for 15 minutes, take the supernatant, use the BAC protein quantification kit to quantify the total protein content of each tissue, and then carry out Tricine SDS-PAGE electrophoresis by preparing the gel according to the concentration of the stacking gel at 4% and the concentrati...
Embodiment 3
[0025] Example 3: Immunohistochemical detection of Clpsl2 protein expression localization in epididymis
[0026] Frozen sections of epididymis tissue were taken and incubated with 3% hydrogen peroxide for 10 min. After blocking with 10% goat serum in a wet box at 37°C for 2-3 hours, incubate overnight in a wet box at 4°C with anti-meClpsl polyclonal antiserum (volume ratio 1:500) prepared with 2% BSA by mass. The next day, they were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (volume ratio 1:300) in a humid box at 37°C for 1 hour. DAB color development, hematoxylin counterstaining gradient alcohol dehydration, xylene transparent, neutral gum mounting, observation under an inverted microscope found that the expression of Clpsl2 protein can be detected in the wall cells of the entire head of the epididymis, and in the head proximal end ( image 3 A, F) The expression level was significantly higher than that in the distal end ( image 3 B), there is a sm...
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