Application of ARL16 protein agonist in sperm flagellum multiple morphological abnormality under hypoxia
A morphological abnormality and agonist technology, applied in the field of DNA recombination, can solve the problems of poor treatment effect, unclear genetic etiology and pathogenic mechanism of HMMAF, etc., and achieve the effect of improving exercise ability and morphological abnormality.
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Embodiment 1
[0033] Example 1 Variation of Arl16 in low oxygen mouse sperm
[0034] 1. Preparation of mouse hypoxic HMMAF model: mouse is placed in an simulated sea level 5800m plateau low pressure oxygen chamber (pressure: about 360 mmHg, oxygen content 10%), and continuous low-pressure hypoxic treatment 10W to replicate mouse low oxygen HMMAF models. The copy of the low oxygen HMMAF model was identified. The 21% oxygen concentration was set to 10 weeks in the control group.
[0035] 2, sperm collection: Open the abdominal cavity, take out the epididymis tissue, extract the bilateral epididellation tailings, and obtain sperm after liquefaction and cleavage. Western blot detects the expression of Arl16 in the test of the test of the test. The specific method is as follows:
[0036] (1) Preparation of protein
[0037] 1) Rinse the cells to be treated with a 37 ° C warm bath, add the pre-cooling lysate (add PMSF, 1 mm to the final concentration), scratch the cells under ice, transfer to the ice ...
Embodiment 2
[0057] Example 2 Overgramps ARL16 vector construction
[0058] 1, primer design
[0059] The genomic sequence of mouse ARL16 genes is GenBank Accession No. Nm_197995.2 (Updatedate 2020-03-25), which is designed according to the genome sequence, and the primer sequence used is as follows:
[0060] Positive primer (SEQ ID NO.2): 5'-ctgatgcgggaagaac-3 '
[0061] Reverse primer (SEQ ID NO.3): 5'-TTGCCCCCCCCA-3 '
[0062] 2, PCR amplification target fragment
[0063] The mouse genomic DNA was extracted, and the PCR amplification system shown in Table 2 was formulated, gently mixed, placed in the PCR instrument, and responded according to the PCR program shown in Table 3;
[0064] Table 2 PCR amplification system
[0065]
[0066] Table 3 PCR program
[0067]
[0068] 3, ARL16 gene fragment and carrier connection
[0069] (1) Padeasy adenovirus expression vector (purchased from Jikaii)
[0070] The reagent was sequentially added in the order in Table 4, mixed, and placed in a 37 ° C...
Embodiment 3
[0092] Example 3 Adenovirus Packaging
[0093] 1, cell culture
[0094] One day before transfection, 293 cells were seeded in 60 mm petri dishes, and the medium was DMEM + 10% HYCLON fetal bovine serum, and the 37 ° C containing 5% CO was placed. 2 The incubator was cultured overnight.
[0095] 2, transfection
[0096] When the cells are grown to the bottom area of 70 ~ 80%, the recombinant adenoviral carrier linearized plasmid is taken, using LiPofiter. TM Transfection reagents were transfected. The specific steps are:
[0097] 1) Replace the complete medium for 2 hours before transfection, and 4 μg of recombinant adenoviral carrier linearized plasmid was touched with 300 μl of DMEM culture solution, and the room temperature was placed for 5 min.
[0098] 2) Take 15 μL of LiPofiter TM Dilution was carried out with 300 μl of DMEM culture solution, and it was placed at room temperature for 5 min.
[0099] 3) Mix the two to prevent light for 20 min. The mixture was then added to a...
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