Method for preparing atherosclerosis disease canine model
A technology for models and female dogs, which is applied in the field of preparing apolipoprotein E gene knockout disease model dogs using gene knockout technology, can solve the problems of no relevant reports, etc., and achieve the effect of improving utilization efficiency and increasing the number
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Embodiment 1
[0053] Example 1: Construction, in vitro transcription and verification of transgene targeting vector
[0054] According to the canine APOE gene sequence information provided by NCBI, the targeting site sequence 5'-CCGGGTGGCAGACTGGCCAGCCC-3' (SEQ ID NO: 4) was selected based on the canine APOE gene Exon3 (see figure 1 ), the sgRNA sequence recognizing this site is 5'-GGGCTGGCCAGTCTGCCACC-3' (SEQ ID NO: 10). When constructing the vector, the backbone carrier T7-gRNA (purchased from Addgene) was digested with BbsI for subsequent experiments; the sgRNA sequence was designed: ataGGGCTGGCCAGTCTGCCACCgt (SEQ ID NO: 5) and the sgRNA complementary sequence: taaaacGGTGGCAGACTGGCCAGCC (SEQ ID NO: 6 ); the sgRNA sequence and the sgRNA complementary sequence are annealed, and then ligated with the digested T7-gRNA plasmid. The T7-sgRNA plasmid was amplified by PCR and the PCR product was recovered, and the PCR product of T7-sgRNA was transcribed in vitro using an in vitro transcription k...
Embodiment 2
[0063] Example 2: APOE gene knockout dog embryo transfer
[0064] A total of 13 beagle bitches in natural estrus were used as fertilized egg donors and embryo transfer recipients. Blood was collected from all bitches to detect the concentration of progesterone in the serum. When the concentration of progesterone reached 4-7ng / mL, it could be determined as the ovulation period. Natural mating was carried out 48 hours after ovulation, and then the fertilized embryos were washed out. 13 bitches were accumulatively fertilized 65 eggs. After collecting the fertilized eggs, use TCM199 medium containing 0.1% hyaluronidase to remove cumulus granulosa cells, then put them into microdrops of HEPES-buffered TCM199 medium (HM, GIBCO11150), and then place them in a micromanipulator equipped with a micromanipulator. instrument on an inverted microscope. Use the microinjection needle to draw the mixed solution containing the mRNA of the sgRNA prepared in Example 1 and the mRNA of Cas9 at a...
Embodiment 3
[0070] Example 3: Detection of gene mutations in APOE knockout dogs
[0071]After puppies were born, ear tissues and tail tissues were collected for identification. After the tissue pieces were shredded in the centrifuge tube, proteinase K was added to a water bath at 56°C for 1-3 hours. Then use a pipette gun to draw 700 μL of Genomic Lysis Buffer, add to the lysis system, mix evenly by inverting up and down, centrifuge at 10,000 g for 1 min. Use a pipette to draw the supernatant to the purification column, 10000g, let stand at room temperature for 1min, and centrifuge for 1min. Replace with a new collection tube, add 200 μL of DNA Pre-Wash Buffer, 10000 g, to the spin column, let stand at room temperature for 1 min, centrifuge for 1 min, and discard the waste. Add 400 μL of g-DNA Wash Buffer, 10000 g, to the spin column, let stand at room temperature for 1 min, centrifuge for 1 min, and discard the waste. Re-centrifuge the purification column and collection tube at 10000g...
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