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Method for constructing genetic character chimeric multi-cellar structure

A structural method and a technology for genetic traits, which are applied in the field of constructing multicellular structures chimeric with different genetic traits, can solve the problems of long time and low efficiency, and achieve the effects of simple method, shortened operation time and improved efficiency.

Inactive Publication Date: 2014-12-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Using traditional means to create genetic chimeras for research purposes requires more and complex genetic manipulations on individual organisms, which is time-consuming and inefficient

Method used

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  • Method for constructing genetic character chimeric multi-cellar structure
  • Method for constructing genetic character chimeric multi-cellar structure
  • Method for constructing genetic character chimeric multi-cellar structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The MDCK cell line is used as the basic cell, and green fluorescent protein and red fluorescent protein are respectively expressed in two different cells.

[0029] Simply mix the above two kinds of cells expressing green and red fluorescent proteins at a ratio of 10:1, and the results after culturing for 96 hours are as follows: figure 1 As shown, panel A is a photo of detecting green fluorescent protein, panel B is a photo of detecting red fluorescent protein, and panel C shows the state of all cells growing in a two-dimensional plane. Simple mixing of the two types of cells did not produce a three-dimensional multicellular structure. This embodiment is a comparative example.

Embodiment 2

[0031] The MDCK cell line was used as the basic cell, and the green fluorescent protein was expressed in the MDCK cells, and the expression positions of the green fluorescent protein were located in microfilaments and nuclei, respectively. In the preliminary experiment, immunofluorescence was performed on ordinary MDCK cell lines grown in two-dimensional planes fixed with paraformaldehyde and MDCK cell colonies expressing green fluorescent protein (cells cultured separately in two-dimensional dispersion and cells in dense culture in two dimensions) Staining and detection of E-Cadherin protein, it can be observed that there is an obvious enrichment of fluorescent signals between densely cultured cells, and it can be confirmed that there is a difference between the above-mentioned common MDCK cells and MDCK cells expressing green fluorescent protein. Obvious adhesive junctions.

[0032] Suspend the MDCK cells expressing green fluorescent protein and ordinary MDCK cells respectiv...

Embodiment 3

[0039] The MDCK cell line is used as the basic cell, and green fluorescent protein and red fluorescent protein are respectively expressed in two different cells.

[0040] Immunofluorescence staining was performed on the above-mentioned cell population and common MDCK cell lines to detect E-Cadherin protein. For MDCK cells with obvious intercellular junctions, the method in Example 2 can be used to promote cell junctions, or the following nucleic acid can be used to label cells Methods.

[0041] If E-Cadherin protein is not detected, there is no obvious intercellular connection between these MDCK cells, and the following nucleic acid labeling methods are used to promote cell connection:

[0042] (1) Prepare nucleic acid single strands complementary to the sequence that can label cells, and prepare complementary sense strands and antisense strands respectively, the length of which is 20 to 30 bases, the single strand has no secondary structure, and the GC content is 50%. Single...

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Abstract

The invention relates to the biotechnical field, and discloses a method for constructing a genetic character chimeric multi-cellar structure. The method comprises the following steps: carrying out immunofluorescence staining on cell communities with different genetic backgrounds and needing chimericity, and observing to determine whether the above cells have intercellular junctions or not; mixing the cells with the intercellular junctions, adding a culture solution, placing the obtained product in a low protein adsorption centrifuge tube, allowing the centrifuge tube to stand at room temperature for 10-20min in order to make the cells precipitate to the bottom of the centrifuge tube, taking out the cells and the culture solution, placing the obtained product in a container, and growing to form a three dimensional structure with functions; and respectively labeling the cells with different genetic backgrounds, having no intercellular junctions and needing junctions by complementary nucleic acid single strands connected with NHS, mixing the labeled cells, placing the obtained product in the container, and growing to form the three-dimensional structure with functions. The method has the advantages of operation time shortening, simplicity, efficiency improvement and good chimeric effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to cell culture, in particular to a method for constructing a chimeric multicellular structure with different genetic traits. Background technique [0002] Biological chimerism refers to individuals who are genetically chimeric or mixed with different genetic traits. Genetic chimera is a very effective tool for studying gene function. In the same environment, the same body, and different parts of the same organ are composed of cells with different genetic backgrounds, so that the effects of differential genes in the organ can be compared. [0003] The multicellular structure in vitro is different from the culture of single cells. Multiple cells can interact in a suitable growth environment in vitro to form functional structural units, which partially mimic the functions of their source tissues in vivo. [0004] To create a multicellular structure with chimeric genetic traits, it is ne...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 蔡亮
Owner FUDAN UNIV
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