Construction and application of transgenic mouse model for expressing farnesyl pyrophosphate synthetase
A technology of nickle pyrophosphate, transgenic mice, applied in the direction of using microinjection method, preparations for in vivo experiments, other methods of inserting foreign genetic material, etc., can solve ventricular vascular remodeling, increase Alzheimer's disease Neuropathological changes, embryonic lethality, etc.
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Embodiment 1
[0029] Example 1 Construction of recombinant expression vector pJG-mFPPS
[0030]Using the cDNA obtained by reverse transcription of the total RNA of the brain tissue of C57BL / 6 mice as a template, the FPPS gene fragment was obtained by conventional RT-PCR method. The primers used in PCR were: P1:5'-ATATGCTAGCGCTACCGGTCGCCACCATGAATGGGAACCAGAAATTGG -3' and P2: 5'-ATTGGATCCTCACTTTCTCCGTTTGTAGATC-3'. After the PCR product was digested with NheI and BamHI, it was inserted into the plasmid pEGFP-C1 digested with NheI and BamHI to generate a new plasmid pE-mFPPS (the FPPS gene cDNA sequence replaced the EGFP sequence in pEGFP-C1, and the mouse FPPS gene cDNA sequence: aaaaagtggctcgggtgaaagcactgtatgaggcgctggatctgcagtctgctttcttcaagtatgaggaagacagttacaaccgcctcaagagtctcatagagcagtgctctgcgcccctgcccccatccatcttcatggaacttgcaaacaagatctacaaacggagaaagtga)在质粒pE-mFPPS中用以下引物:P3:5'-ACGCGTCGACATGAATGGGAACCAGAAATTG-3'和P4:5'-CCCAAGCTTTCACTTTCTCCGTTTGTAGATCTTG-3'扩增出FPPS cDNA片段,经SalI及HindIII酶切插入pJG / a...
Embodiment 2
[0031] Example 2 Preparation and identification of transgenic mice
[0032] After the pJG-mFPPS plasmid was linearized by restriction endonuclease BamHI, electrophoresis, gel extraction kit (Qiagen, CA) was used to purify and recover the 7.1kb fragment, dissolved in TE, and adjusted the final concentration to 2ng / μl. By microinjection, the injected oocytes are transplanted into the fallopian tubes of pseudopregnant mice, and the mice are delivered after about 20 gestations.
[0033] Identification of the first transgenic mouse (G0) by PCR method: snip the tail of the 10-day-old mouse, extract the genomic DNA, and use primers (P5: 5'- ACGCGTCGACATGAATGGGAACCAGAAATTG -3'; P6: 5'- GCCTGGAATCCCAACAAC -3' ), carry out PCR reaction, positive mouse can produce about 1.4kb fragment ( Figure 4 ).
[0034] In order to study the passage of the transgene in transgenic mice, we crossed the first transgenic mice with normal C57BL / 6 mice to generate the first generation of transgenic m...
Embodiment 3
[0036] Example 3 Expression of transgenes in mice
[0037] According to the kit instructions, RNAiso reagent (TaKaRa, DL) was used to extract total RNA from heart tissues of normal and transgenic mice. After digestion with DNaseI (TaKaRa), reverse transcription (TaKaRa) produced the first-strand cDNA. Real-time PCR reaction was performed on this cDNA, the internal reference β-actin primers were P7: 5'-CGTCAGATCCGCTAGCGCTACC-3', P8: 5'-CCGGCGAGTGAGGGAAGAGTC-3', and the FPPS primers were P9:5-GGAGGTCCTAGAGTACAATGCC-3', P10 5'-AAGCCTGGAGCAGTTCTACAC-3'. The PCR reaction conditions are: 95°C pre-denaturation for 2min, 95°C for 10s, 60°C for 40s, 40 cycles.
[0038] According to the instructions (Santa Cruz Biotechnology, Inc. CA), protein samples from tissues were prepared for Western blotting. Load 30 μg of each sample, 12% SDS-PAGE electrophoresis, transfer to PVDF membrane, 5% skimmed milk powder (TBST) to block for 1 hour, anti-FPPS antibody (1:1500, Clontech) and anti-β-a...
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