Alzheimer’s Disease Modeling

Alzheimer’s Disease (AD), a neurodegenerative disease, is a disorder characterized by a progressive decline in cognitive functions and loss of specific types of neurons and synapses[1]. The most recognized pathological events are amyloid plaques and neurofibrillary tangles (NFTs) consisting of amyloid-β peptide (Aβ) and hyperphosphorylated tau protein in AD^[2]. Alzheimer's symptoms can be simulated in mice by injecting drugs (such as Aβ) or genetically modified.

MCE has not independently verified the accuracy of these methods. They are for reference only.

I． Aβ Injection Mice Model

1. Prepared Aβ solution: Aβ was dissolved in sterile normal saline, diluted, divided into frozen storage in the refrigerator for use (incubate in an incubator before use).

2. Anesthesia: Before preparing for surgery, used isoflurane to anesthetize mice or intraperitoneal injection of chloral hydrate.

3. Fixed mouse: Fixed mouse head on brain stereoscopic locator, prone position. The hair on the top of the mouse head was cut off with scissors, and the skin was cut open with a scalpel after disinfection with iodophor.

4. Localization injection: bilateral hippocampal CA1 region was selected as the directional injection area, marked the injection point, and the needle was vertically injected into the dura surface with a micro sampler at a depth of 2.0 mm. The prepared Aβ solution was slowly injected, the needle was left for 2 minutes after the injection to ensure that the solution was fully dispersed into the local hippocampal tissue, and then the needle was slowly withdrawn.

5. Suture: Closed the wound, sutured the incision, used gentamicin injection to reduce inflammation at the suture, and the modeling was completed.

II． Transgenic Mouse models

1. Generation of Tg2576 Mice Model^[2][3][4]

Tg2576 expresses the mutated AβPP 695 driven by the PrP promoter.

a). Constructed the plasmid PrP-hAPP695 with neuron-specific promoter.

b). Injected the transgenic plasmid into the fertilized eggs of C57BL/6J mice by microinjection.

c). The injected embryos were reimplanted into foster mothers.

d). Screening and identify the resulting offspring genotyped to obtain Tg2576 mice.

2. Generation of APP/PS Mice Model^[4]

a). Crossed C57BL/6J mice that express PrP-hAPPK595N/M596L (mutant APP) and PrP-hPS1ΔE9 (mutant PSI), respectively.

b). Screening and identify the resulting offspring genotyped to obtain APP/PS mice.

3. Generation of 5×FAD Mice Model^[5]

The 5×FAD mouse model combines the Swedish mutation (K670N, M671L) at the ß-cleavage site with the Florida (V717I) and London mutations (I716V) at the y-cleavage site of AβPP, and two additional mutations within the PSEN1 gene (M146V and L286V), both transgenesunder the control of the murine Thy-1 promoter. 5×FAD model exhibits AD pathology.

a). Introduced the FAD mutations APP (K670N/M67IL + I716V+V717I) and PS1 (M146L+L286V) into APP (695) and PS1 cDNAs by site-directed mutagenesis. (1716V)

b). Then subcloned into exon 2 of the mouse Thyl transgene cassette.

c). Sequenced following standard procedures.

d). After purification of the transgenes from vector sequence, the transgenes were added together in equal proportions and microinjected into the pronuclei of single-cell C57/B6XSJL hybrid embryos.

e). Identified and screened founder transgenic mice.

f). The three highest APP and PS1 transgene coexpressing lines were bred with B6/SJL F1 hybrids for analysis screening to obtained stably expressing 5×FAD transgenic mice.

4. Generation of 3xTg-AD Mouse Model^[6]

The tripletransgenic mouse model of AD (3×Tg-AD), which combine AßPPSwe, PS1M146V, and taup3o1L human mutations and develops both amyloid plaques and NFTsin AD relevant regions.

a). Using the pronuclear microinjection technique, coinjected two independent transgene constructs encoding human APPswe and taup31L (4R/ON), both under the control of the mouse Thy1.2 regulatory elements, into single-cell embryos harvested from mutant homozygous PS1M146v knockin mice.

b). The injected embryos were reimplanted into foster mothers.

c). Screening and identify the resulting offspring genotyped to obtain 3×Tg-AD mice.