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Usage of AAV-DJ type adeno-associated virus in-vitro efficient infection organoid

An AAV-DJ, organoid technology, applied in the field of genetic engineering and cell engineering, can solve the problem of less research and achieve the effect of efficient infection

Active Publication Date: 2018-12-25
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few studies on the efficient infection of organoids by AAV in vitro. Therefore, providing a method for efficient infection of organoids by AAV in vitro has become an urgent problem to be solved at this stage.

Method used

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  • Usage of AAV-DJ type adeno-associated virus in-vitro efficient infection organoid
  • Usage of AAV-DJ type adeno-associated virus in-vitro efficient infection organoid
  • Usage of AAV-DJ type adeno-associated virus in-vitro efficient infection organoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Isolation and culture of liver organoids

[0032] For the isolation and culture of liver organoids, refer to the method reported in [Broutier, et al. (2016). Culture and establishment of self-renewing human and mouse adult liver and pancreas 3 Dorganoids and their genetic manipulation. Nature protocols 11, 1724-1743.] , Liver organoids were isolated from liver tissues of wild-type C57 / B6 mice and humans for virus infection. The specific methods are as follows:

[0033] 1. Immediately after the mouse was euthanized, the liver was taken out, and placed in the 4-degree pre-cooled basal medium, the basal medium components were: Advanced DMEM / F12 medium, penicillin, GlutaMax and HEPES;

[0034] 2. Cut the liver to 0.5mm with surgical scissors 3 Transfer it to a 15ml centrifuge tube, add 10ml washing medium (composition: DMEM medium, 1% fetal bovine serum, 1% penicillin) and beat repeatedly;

[0035] 3. Allow to settle and remove the supernatant. Then add 10ml o...

Embodiment 2

[0044] Example 2: Isolation and culture of intestinal organoids

[0045] Isolation and culture of small intestine organoids refer to the method reported in [Sato, et al. (2009). Single Lgr5 stemcells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265.], from wild-type Small intestinal organoids were isolated from C57 / B6 mouse and human liver tissues for virus infection. The specific method is as follows:

[0046] 1. After the mice were euthanized, the small intestines were taken out and placed in 4-degree pre-cooled PBS buffer;

[0047] 2. Cut the small intestine longitudinally with surgical scissors, and rinse with PBS buffer 3 times to remove chyme;

[0048] 3. Use a scalpel to carefully scrape off the small intestinal villi on the inner wall of the small intestine;

[0049] 4. Cut the remaining part into 5mm length intestinal slices and incubate on ice with PBS buffer containing 10mM EDTA for 40 minutes;

[0050] 5. Replace the PBS bu...

Embodiment 3

[0053] Example 3: AAV-DJ virus infects organoids (Here, liver organoids are taken as an example, small intestine and kidney organoids are operated similarly)

[0054] 1. Separate the organoids in 3D culture together with Matrigel from the well plate, rinse the well plate once with PBS, and collect it in a 1.5ml centrifuge tube;

[0055] 2. Centrifuge at 900rpm for 1min and remove the supernatant;

[0056] 3. Add 1ml of PBS buffer solution, blow and mix well to remove the matrigel around the organoids;

[0057] 4. Centrifuge at 1000 rpm for 1 min and remove the supernatant;

[0058] 5. Spread 40 μl of pre-cooled Matrigel evenly in a 24-well plate, and let it solidify for 10 minutes;

[0059] 6. Dilute AAV-DJ virus with 400 μl organoid medium, according to MOI=10E6 (the higher the MOI, the higher the infection efficiency), that is, to infect 10,000 cells, only 1 μl AAV-DJ virus (titer is 10E13 vgs / ml);

[0060] 7. Resuspend the organoid pellet in step 4 with the virus-added ...

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Abstract

The invention belongs to the technical field of the genetic engineering and cell engineering, and particularly relates to a usage of an AAV-DJ type adeno-associated virus in-vitro efficient infectionorganoid. Commercial AAV-DJ type adeno-associated viruses are used for performing in-vitro planarization infection on the organoids of a human and a mouse, after the infection, the organoids of planarization culture is cultured for 72 hours again by using a 3D culture system. Finally, efficiency of infection of the organoids is detected by using a fluorescence confocal microphotograph technology in combination with a flow cytometry. It is indicated from a result that the AAV-DJ viruses can efficiently infect the organoids of the human and the mouse in vitro, and the efficiency of infection canreach 80% or more, and is far superior to a reported organoid genetic manipulation tool. The usage solves a big problem for scholars of organoid in-vitro research, and provides a firm foundation forclinical research of hepatic regeneration.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and cell engineering, and specifically relates to an application of an AAV-DJ type adeno-associated virus to efficiently infect organoids in vitro. Background technique [0002] Organoids are a type of cell clusters that are close to physiological conditions in vivo, have self-renewal and self-organization capabilities, and have corresponding tissue and organ functions. Organoids are derived from primary tissues, one of embryonic stem cells and induced pluripotent stem cells, and rely on artificial extracellular matrix (ECM) to self-organize and restore the original tissue cell structure. Unlike traditional in vitro cultures, organoids have similar cellular composition and structure to primary tissues, and contain a small fraction of stem cells with stable genomes, self-renewal and differentiation potential. Another important feature is that organoids can be expanded in vitro indefini...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00
CPCC12N5/0671C12N7/00C12N2501/11C12N2501/119C12N2501/12C12N2501/345C12N2501/415C12N2750/14131
Inventor 赵冰魏劲松
Owner FUDAN UNIV
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