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Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof

A non-human mammal, neuropsychiatric disease technology, applied in animal cells, vertebrate cells, pharmaceutical formulations, etc., can solve the problems of unsatisfactory neuropsychiatric disease animal models and complex causes.

Active Publication Date: 2015-03-25
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex causes of diseases such as schizophrenia, so far, no satisfactory animal models of neuropsychiatric diseases have been obtained by introducing or knocking out so-called "important candidate genes for schizophrenia"

Method used

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  • Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof
  • Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof
  • Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Example 1 Construction of Targeting Vector

[0161] 1.1 Extraction of mouse BAC DNA

[0162] Streak the BAC (RPCI23-414A17) DH10B bacteria containing the full-length CRMP2 genome on a plate containing chloramphenicol, culture at 37°C for 10 hours, pick 5 single clones, and shake 5 mL of bacteria respectively for 12 hours. 13000rpm, 1min to collect the bacteria, add 300μL P1 (Tris50mM, EDTA10mM, pH8.0) to suspend the bacteria, then add 300μL P2 (0.2M NaOH, 1%SDS), mix well, leave at room temperature for 5min, then add 300μL P3 (3M KAC, pH5.5), shake gently for 2-5min, centrifuge at 10000rpm, 4°C for 10min. Transfer the supernatant to a new EP tube, then add 800 μL of pre-cooled isopropanol, mix upside down and place on ice for 5 minutes, 4°C, 10,000 rpm, 15 minutes, remove the supernatant, wash the precipitate twice with 600 μL of 70% ethanol, and dry in the air , add 40 μL double distilled water, and 1 μL RNaseA, dissolve at 37°C for 30 min.

[0163] 1.2 Preparation ...

Embodiment 2

[0187] Example 2 CRMP2 gene knockout

[0188] 2.1 Gene targeting

[0189] Day 1: Prepare targeting vector, prepare 100mm trophoblast cells

[0190] Target carrier preparation:

[0191] Use NotI to linearize 200 μg of the targeting vector (plasmid extraction method according to QiaGen EndoFree Plasmid Maxi Kit), add twice the volume of absolute ethanol, place at -80°C for 30 minutes, 12000 rpm, 30 minutes, recover by centrifugation, wash twice with 70% ethanol, and dry in the air. Dissolve in 50 μL Milli-Q water.

[0192] Primary mouse fibroblast (MEF) cell recovery culture:

[0193] The cryopreserved MEF cells were taken out from liquid nitrogen, and the MEF feeder layer cells were quickly thawed at 37°C, and the MEF medium was added to a total volume of 10 mL, and the cells were collected by centrifugation at 1000 rpm for 5 min. Resuspend the cells with 10mL MEF medium and plant them on a 100mm Petri dish.

[0194] Day 2: Check trophoblast cells and recover ES cells

[...

Embodiment 3

[0253] The preparation of embodiment 3 animal model

[0254] 3.1 Acquisition of CRMP2 chimeric mice and F1 generation mice

[0255] The identified positive ES cell clones were revived for blastocyst microinjection to inject a total of 90 blastocysts, each embryo was injected with 15 ES cells, and then 90 embryos were transplanted into the uterus of 8 ICR pseudopregnant mother mice. After 19 days, 18 daughter mice were produced, and 10 chimeras survived, of which 6 were male mice, such as Figure 2-6 As shown, only one has a chimerism of about 30%, and the remaining five are all gray. The obtained 6 male mice were mated with C57BL / 6J female mice respectively. Among them, 2 gray mice were infertile, and the other 4 mice were fertile ( Figure 5 ).

[0256] 3.2 Breeding strategy of CRMP2 conditional knockout mice

[0257] The positive CRMP2flox / + born of chimeric male mice mated with C57BL / 6J female mice was defined as the F1 generation. F1 CRMP2flox / + female mice were mate...

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Abstract

The invention relates to a non-human mammal animal model of neurological and psychiatric diseases and a preparation method and application thereof. A mouse model provided by the invention is in high similarity with the model of human neurological and psychiatric diseases. A neurological and psychiatric diseases drug screening platform based on the animal model can be used in new drug screening and development of other treatments.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a non-human mammalian neuropsychiatric disease animal model and its preparation method and application. Background technique [0002] Schizophrenia is the most common type of mental illness, affecting 0.5%-1% of the world's population, characterized by basic personality changes, split thinking, emotion and behavior, and incoordination between mental activities and the environment. The disease mostly occurs in men from late puberty to 25 years old, and in women 25-35 years old is the high incidence period. The natural course of the disease is often protracted, and most of them are in the process of relapse and aggravation, chronicity and decline, and are genetically related to other mental diseases. The nature of the disease is strong, the overlap rate is high, and the final outcome is that about half of the patients are mentally disabled an...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/09A01K67/027A61K49/00
Inventor 许执恒
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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