Slide climbing method of PGCs (primordial germ cells)

A technology of primordial germ cells and slices, which is applied in the biological field, can solve time-consuming and labor-intensive problems, and achieve the effects of saving time, related expenses, and cost

Active Publication Date: 2014-03-26
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing primordial germ cell climbing method is as follows: put the glass slide in a petri dish, add feeder cells, after the feeder cells grow into a single layer on the glass slide, use mitomycin or γ-ray After the treatment terminates its division, add PGCs cells, after about 24 hours of cultivation,

Method used

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  • Slide climbing method of PGCs (primordial germ cells)
  • Slide climbing method of PGCs (primordial germ cells)
  • Slide climbing method of PGCs (primordial germ cells)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: a kind of climbing method of primordial germ cells (such as figure 1 shown)

[0029] 1) Primordial germ cells are purified by Nycodenz density gradient centrifugation, Percoll density gradient centrifugation or flow sorting;

[0030] 2) Wash the purified primordial germ cells 3 times with PBS, and collect the cells by centrifugation at 200 g for 5 minutes;

[0031] 3) Pick 100-200 cells under a microscope and wash them once in PBS droplets on a glass slide;

[0032] 4) Pick the cells and wash once in a 4% paraformaldehyde solution;

[0033] 5) Pick the cells and fix them in 4% paraformaldehyde droplets on the slide for 10-30 minutes;

[0034] 6) Absorb the paraformaldehyde with a concentration of 4%, and complete the climbing and fixing of PGCs cells.

[0035]

Embodiment 2

[0036] Example 2: AKP (alkaline phosphatase) staining of PGCs cells:

[0037] According to Example 1, slice and fix the PGCs cells, remove the 4% paraformaldehyde solution, wash 3 times with 200 μL PBS, 5 minutes each time; add BCIP / NBT staining solution, and incubate in a humid environment at room temperature for 1 h; wash After slicing, observe and take pictures under an inverted microscope.

[0038] Such as figure 2 As shown, because PGCs cells are rich in alkaline phosphatase, the stained PGCs cells appear blue.

[0039]

Embodiment 3

[0040] Example 3: PAS (alkaline phosphatase) staining of PGCs cells:

[0041] Slice and fix PGCs cells according to Example 1, remove 4% paraformaldehyde solution, wash 3 times with 200 μL PBS, 5 minutes each time; add Periodic Acid Solution, incubate at room temperature for 5 minutes; add Schifff's Reagent, incubate at room temperature for 15 minutes , After washing, observe under an inverted microscope and take pictures.

[0042] Such as image 3 As shown, because PGCs cells contain a lot of glycogen, they are red after PAS staining.

[0043]

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Abstract

The invention discloses a slide climbing method of PGCs (primordial germ cells) and belongs to the biotechnology field. The slide climbing method comprises the steps as follows: purifying the PGCs; washing the purified PGCs with a PBS (phosphate buffer solution) for three times, and centrifugally collecting the cells; selecting and putting 100-200 cells in PBS droplets on a glass slide under a microscope for washing for one time; selecting and putting the cells in a paraformaldehyde solution with the concentration of 4% on the glass slide for washing for one time; selecting and putting the cells on paraformaldehyde droplets with the concentration of 4% on the glass slide for fixation for 10-30 min; and removing the paraformaldehyde with the concentration of 4% through adsorption, and finishing slide climbing of the cells while fixing the cells. According to the slide climbing method, the problem that the purified PGCs are difficult to climb the glass slide is solved, and the time and the cost are saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for climbing primordial germ cells. Background technique [0002] Primordial germ cells (PGCs) are precursor cells that form germ cells, which can specifically generate sperm and eggs in sexually mature individuals (Durcova-Hills G et al., PloS one, 2008.3: e3531). As an oviparous animal, poultry is difficult to obtain a single embryo, so the research on PGCs cells is particularly important. With the establishment of the PGCs cell in vitro culture system, the research on modern conservation technology and transgenic technology based on PGCs cells has developed rapidly. [0003] PGCs cells are an ideal raw material for preparing transgenic animals. In 2006, Marie-Cecile van de Lavoir and others successfully established the long-term culture technology of chicken PGCs cells in vitro. The PGCs cells cultured in vitro for about 200 days still have the characteristic...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/30
Inventor 于福先潘建治朱志伟陈晓宇黄菁
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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