CN-121975868-A - Construction method and application of Rosa 26-APP-eta-CTF transgenic mouse model

Abstract

The invention discloses a construction method and application of a Rosa 26-APP-eta-CTF transgenic mouse model. The invention utilizes CRISPR/Cas9 mediated embryo gene editing technology, inserts the expression cassette containing the humanized APP-eta-CTF-mCherry fusion gene into the mouse Rosa26 site at fixed points through homologous recombination repair mechanism, and constructs the inducible expression transgenic mouse model. The model fills the blank of the prior art lacking an animal model for specifically simulating the accumulation of eta-CTF in vivo, and provides a key tool for researching the action of eta-secretase in Alzheimer disease.

Inventors

• XIE YUHUAN
• ZHU YOUYANG
• GUO PEIXIN

Assignees

• 云南中医药大学

Dates

Publication Date

20260505

Application Date

20260326

Claims (4)

1. 1. The method for constructing the Rosa 26-APP-eta-CTF transgenic mouse model is characterized by comprising the following steps of: (1) Constructing a targeting vector comprising a5 'homology arm, a CAG-Pr, an rtTA coding sequence, 4 XpA, a TRE-Pr, a cDNA coding for CTF-eta, which is connected with a fluorescent protein mCherry coding sequence through a P2A sequence, and is connected with a WPRE element, pA and a 3' homology arm in sequence at the downstream, wherein: The sequence of the 5' homologous arm is shown as SEQ ID NO. 1; the cDNA sequence for coding CTF-eta is shown as SEQ ID NO. 2; the sequence of the 3' homologous arm is shown as SEQ ID NO. 3; Meanwhile, the sgRNA is designed, and the sequence is shown as SEQ ID NO. 4; (2) The construction of the mice comprises the steps of mixing a targeting vector, sgRNA and Cas9 mRNA, introducing the mixture into fertilized eggs of C57BL/6 mice through microinjection, transplanting the fertilized eggs after injection into pseudopregnant female mice to obtain F0-generation mice, carrying out genotype identification on the F0-generation mice through PCR and sequencing, and backcrossing the positive construction mice with wild C57BL/6 mice to obtain genotype-stable F1-generation heterozygote mice.
2. 2. Use of a Rosa26-APP- η -CTF transgenic mouse model obtained by the construction method of claim 1 for the preparation of a product for studying the pathogenesis of AD.
3. 3. Use of a Rosa26-APP- η -CTF transgenic mouse model obtained by the method of construction according to claim 1 for the preparation of a product for finding a therapeutic target for AD.
4. 4. Use of a Rosa26-APP- η -CTF transgenic mouse model obtained by the method of construction according to claim 1 for the preparation of a product for evaluating the efficacy of a medicament for the treatment of AD.

Description

Construction method and application of Rosa 26-APP-eta-CTF transgenic mouse model Technical Field The invention belongs to the technical field of biology, and particularly relates to a Rosa 26-APP-eta-CTF transgenic mouse model constructed by using a CRISPR/Cas9 mediated embryo gene editing technology, which is used for simulating a pathological mechanism of eta-CTF accumulation in Alzheimer disease and is used for drug screening and cognitive dysfunction research. Background Alzheimer's Disease (AD) is one of the most common types of dementia, and is characterized by progressive learning and memory dysfunction as a major clinical feature, belonging to complex neurodegenerative diseases. Its pathogenesis involves a number of factors including age-related changes, amyloid Precursor Protein (APP) and presenilin gene mutations, genetic risk, vascular disease, traumatic Brain Injury (TBI), dietary structure, immune dysfunction, mitochondrial dysfunction, heavy metal exposure and infection, and the like. Among them, the association of TBI with AD is of increasing interest. TBI, a common public health problem, is caused by head or body impact, which can trigger a molecular pathway that has not yet been fully elucidated, promoting AD pathogenesis. The key pathological feature is the rapid accumulation of beta-amyloid (aβ) in the cerebral cortex, about one third of short-term TBI patients develop aβ deposition after injury, and aβ can persist for years after a single injury, suggesting that TBI may accelerate AD progression. Currently, the mainstream hypothesis suggests that TBI induces AD-like pathological changes by promoting aβ accumulation. The production of Abeta results from aberrant metabolism of APP by cleavage of APP by beta-secretase (BACE) to form sAPPbeta and beta carboxy-terminal fragment (beta-CTF) which in turn is hydrolyzed by gamma-secretase to produce Abeta and APP cytoplasmic domain (AICD) in the amyloid pathway. The non-amyloid pathway then cleaves APP by α -secretase to generate sappα and α carboxy-terminal fragments (α -CTF), further generating P3 and AICD. Recent studies have found that the third APP hydrolysis pathway, η -secretase, cleaves APP to produce sAPP η and an Aβ -containing carboxy-terminal fragment (η -CTF), which is further processed by α or β -secretase to produce Aη - α and Aη - β. Although the neurotoxicity of Aβ in AD has been widely demonstrated, the role of the eta-secretase pathway and its products (eta-CTF, A eta-alpha, A eta-beta) is not yet defined. Studies have shown that selective inhibition of β -site amyloid precursor protein lyase 1 (BACE 1) can lead to acute production of a eta-alpha, possibly involving inhibition of long-term potentiation (LTP), affecting synaptic plasticity. Laser capture microscopy showed that eta-secretase(s) and eta-CTF are enriched around amyloid plaques and in atrophic neurites in brain tissue of AD patients, suggesting a potentially important role in AD pathogenesis. However, it is not clear whether accumulation of η -CTF in the brain directly leads to cognitive dysfunction, and the lack of animal models specifically mimicking accumulation of η -CTF in vivo has limited the intensive study of the relevant mechanisms. Disclosure of Invention Aiming at the problem of lack of an animal model for specifically simulating accumulation of eta-CTF in vivo in the prior art, the invention aims to provide a construction method of a Rosa 26-APP-eta-CTF transgenic mouse model and application thereof. The invention utilizes CRISPR/Cas9 mediated embryo gene editing technology, inserts the expression cassette containing the humanized APP-eta-CTF-mCherry fusion gene into the mouse Rosa26 site at fixed points through homologous recombination repair mechanism, and constructs the inducible expression transgenic mouse model. The method comprises the following specific steps: (1) Constructing a targeting vector, wherein the targeting vector comprises a 5 'homology arm (the sequence is shown as SEQ ID NO. 1), CAG-Pr, rtTA coding sequences, 4 XpA, TRE-Pr and cDNA (the sequence is shown as SEQ ID NO. 2) for coding CTF-eta, the targeting vector is connected with a fluorescent protein mCherry coding sequence through a P2A sequence, and a WPRE element, pA and a 3' homology arm (the sequence is shown as SEQ ID NO. 3) are sequentially connected at the downstream; meanwhile, a unidirectional guide RNA (sgRNA) specifically targeting the Rosa26 site is designed, and the sequence of the sgRNA is shown as SEQ ID NO. 4. (2) The construction of the mice comprises the steps of mixing a targeting vector, sgRNA and Cas9 mRNA, introducing the mixture into fertilized eggs of C57BL/6 mice through microinjection, and transplanting the fertilized eggs after injection into pseudopregnant female mice to obtain F0-generation mice. And carrying out genotype identification on the F0 generation mice by PCR and sequencing, and backcrossing the positive established mice with the wild C57BL/6