Alzheimer's disease animal model and use thereof
A wild-type, in vivo technology, applied in applications, animal cells, plant genetic improvement, etc., can solve the problems of neuron loss without neuritic plaques and neurofibrillary tangles
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Embodiment 1
[0328] Example 1: Construction of Donor Nucleic Acid Molecules
[0329] After searching the genomic DNA of rat App from the NCBI database, it was found that the App gene spans about 216.3 kb on the reverse strand of chromosome 11. The gene ID (from the database NCBI) is: 54226. There are two transcripts of this gene (Protein IDs ENSRNOP00000041613 and ENSRNOP00000040243 from Ensembl). The individual domains and features of the rat App gene have been analyzed.
[0330] Then, the rat App genomic DNA fragment was isolated, which included exon 16, intron 16 and exon 17; the fragment including exon 16 and exon 17 was about 4 kb. Then, the Aβ sequence in rat APP was humanized by introducing mutations resulting in G676R, F681Y and R684H amino acid substitutions. In addition, the Swedish double mutation (K670N and M671L) was introduced into exon 16, and the Beyreuther / Iberian (I716F substitution) and Arctic (E693G substitution) mutations were introduced into exon 17. figure 1 A sc...
Embodiment 2
[0332] Example 2: CRISPR construction and activity assay
[0333] In different rat strains, the targeting gene sequence (ie targeting App gene sequence) may be different. Therefore, to ensure the efficiency of Cas9 / sgRNA and to ensure the identity between the sgRNA target and the DNA sequence obtained from the rat tail genome, PCR and DNA sequencing were performed on the genomic DNA obtained from the rat tail. The results are shown in figure 2 , lanes 1 and 2 are the PCR amplification products of the upstream target sequence, lane 3 is the DNA molecular weight label, and lanes 4 and 5 are the PCR amplification products of the downstream target sequence.
[0334] The results showed that the sequences obtained from the PCR products obtained from rat tails were identical to those recorded in the NCBI and Ensembl databases.
[0335] sgRNAs are designed near the insertion site (in the intronic region), and insertions of heterologous fragments containing restriction sites are eas...
Embodiment 3
[0339] Example 3: Injection of Cas9 / sgRNA and donor nucleic acid molecules
[0340] All animal experiments were performed in accordance with the specifications of AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). The IACUC (Institutional Animal Care and Use Committee, Institutional Animal Care and Use Committee) of Tsinghua University approved the animal protocol (15-LB5) used in this study. Rats were maintained on a standard 12 hr light / 12 hr dark cycle and were housed in groups of 1-2. Food and water supplies were not restricted unless otherwise stated.
[0341] The selected single-cell fertilized eggs of the rats were transferred to the prepared M2 medium strips and arranged in a row (about 30-50 eggs / row). Place the injection dish on the stage of an inverted microscope so that the M2 droplet is elongated in the direction perpendicular to the operator, i.e. the y-axis. The in vitro transcribed Cas9 mRNA, sgRNA2 and sgRNA6 (obt...
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