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In-vitro culture system and application of fruit fly retina development model

A technology for in vitro culture and development model, applied in the biological field, can solve the problem of lack of in vitro culture methods, and achieve the effect of rapid preliminary screening

Inactive Publication Date: 2012-10-31
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the early retinal development process cannot be observed in vivo, and there is a lack of effective in vitro culture methods, the details of its real-time development within two hours have not been clarified so far, and the hypothesis about the retinal morphological development process is still only speculation

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  • In-vitro culture system and application of fruit fly retina development model
  • In-vitro culture system and application of fruit fly retina development model
  • In-vitro culture system and application of fruit fly retina development model

Examples

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Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of an in vitro culture system of a Drosophila retinal development model and real-time tracking of developmental progress

[0026] 1 Preparation of organ culture medium

[0027] Inactivate 50ml of fetal bovine serum (FBS, SH30396.03; Hyclone) at 56°C for 30min, cool to room temperature, add 500ml of Schneider's Drosophila medium (11720-034; Invitrogen); add 5ml of penicillin / streptomycin (15140-122 ; Invitrogen), mix evenly, filter and sterilize and store in 4 degrees. Before use, take out an appropriate amount of culture solution, let it stand at room temperature, add 0.2 mg / ml insulin (15500; Sigma), mix well and set aside.

[0028] 2 Construction of a breathable visual platform

[0029]Take a 18mmX18mm cover glass, and evenly spread 10 μl of polychlorotrifluoroethylene (Halocarbon 700 oil, H8898, Sigma) on one side of the cover glass at a distance of 2-3 mm from the four sides. 6 μl of organ culture solution was added dropwise on the air-per...

Embodiment 2

[0032] Example 2: Application of Drosophila retinal development model to screen genes related to early retinal development

[0033] 1. Drosophila containing mutant genes (such as flr EY-P2 ) mated with fruit flies containing a fluorescent marker gene (such as E-cadherin-GFP), and the genotype was constructed as E-cadherin-GFP by genetic methods; flr EY-P2 of fruit flies.

[0034] 2. Isolate the retina of the above-mentioned Drosophila in the organ culture medium. According to the method described in Example 1, a gas-permeable visual platform was built and the retinal development process was tracked and analyzed. Depend on image 3 It can be seen that the single eye of the mutant retina (marked by the dotted line) cannot develop smoothly from the circular cluster to the long-shaped cluster. The position of optic nerve cells 3 and 4 relative to M cells is also difficult to change, so that new cell connections cannot be formed, which will directly lead to defects in later ret...

Embodiment 3

[0035] Example 3: Rapid drug toxicology evaluation using the Drosophila retinal development model

[0036] 1. Add the drug to be screened at a final concentration of 10 μM [such as jasplakinolide (Jasp), J7473, Invitrogen, a drug that disrupts the stability of actin microfilaments) in the organ culture medium, and add an equal amount of solvent dimethyl sulfoxide in one portion (DMSO) organ culture medium was used as a control.

[0037] 2. Isolate the Drosophila retina containing a fluorescent marker (such as E-cadherin-GFP) in the organ culture medium, and firmly seal it in the "gas permeable bottom membrane-cover glass-polychlorotrifluorochloride" according to the method described in Example 1. In the liquid cavity formed by ethylene-organ culture medium containing Jasp or DMSO, the retinal development process was detected by confocal laser microscopy.

[0038] 3. Comparing the retinal development after DMSO control group and Jasp drug treatment, it can be found that after ...

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Abstract

The invention discloses an in-vitro culture and building method of a fruit fly retina development model. The method comprises the steps of: prescription and preparation of a fruit fly cell culture fluid, building and film making of a ventilated visible culture platform, and tracking observation of development, remodeling, splitting, differentiation and the like of retinal cells by using a laser con-focal microscopy. The in-vitro culture system, disclosed by the invention, has the advantages of being rational in design, convenient and fast to operate, capable of culturing retinas of fruit fly larvae in an in-vitro environment for as long as 3 hours and convenient for tracking observation of the early-stage development process of the retina in real time, and provides a beneficial tool for researching the cell and molecular mechanism of the retina development, screening relevant genes and nosogenesis of the retina development, toxicologically evaluating drug screening and screening relevant drugs in an eye disease model.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro culture system of a fruit fly retinal development model, and its application in screening retinal development-related genes and drugs. Background technique [0002] Drosophila is recognized as an excellent model organism by the scientific community due to its simple and cheap breeding method, rapid and large number of reproduction, concise and clear genome for easy research, many genetic methods and easy operation. Since the beginning of the 20th century, Morgan used fruit flies to confirm Mendel's laws and proposed the third law of genetics - "the law of linkage and exchange" and won the Nobel Prize in Medicine and Physiology in 1933 for this, the research based on fruit flies has gradually revealed Molecular regulation mechanism of embryogenesis and organ development. The long-term research results of a large number of scientists have confirmed that most of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12Q1/68C12Q1/02
Inventor 储丹丹陈炯
Owner NANJING UNIV
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