Method for preparing pelochelys biloba chromosome specimen by using peripheral blood cells

A chromosome and blood cell technology, applied in the field of cytogenetics, can solve the problems of scarcity and difficulty, and achieve the effect of easy observation and simple and fast production method

Active Publication Date: 2021-03-30
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the scarcity of the number, the remaining living samples are extremely precious, and it is quite difficult to apply the traditional chromosome specimen preparation method in P.

Method used

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  • Method for preparing pelochelys biloba chromosome specimen by using peripheral blood cells
  • Method for preparing pelochelys biloba chromosome specimen by using peripheral blood cells
  • Method for preparing pelochelys biloba chromosome specimen by using peripheral blood cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Peripheral blood sampling

[0034] Using a commercially available negative pressure blood collection tube (containing sodium heparin) and a sterilized needle, collect 1 ml of peripheral blood from the cervical sinusoids of the live C. After blood collection, it was temporarily stored at 4°C.

[0035] 2. Peripheral blood cell culture

[0036] Cell culture operations were performed under sterile conditions using a modified commercially available Eagle basal medium. The medium modified formula was added on the basal medium, 20mM 4-hydroxyethylpiperazineethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acids, 1mM sodium pyruvate, 1mM penicillin and strepto Mycin double antibody, 55μM β-mercaptoethanol, 15% fetal bovine serum, 5ng / mL recombinant human basic fibroblast growth factor. Add 200 μL of whole blood per 3 ml of medium, and add 750 μg of PHA-P.

[0037] 3. Colchicine treatment

[0038] Blood cells were cultured at 28°C (CO 2 Th...

Embodiment 2

[0042] 1. Peripheral blood sampling

[0043] Using a commercially available negative pressure blood collection tube (containing sodium heparin) and a sterilized needle, collect 1 ml of peripheral blood from the cervical sinusoids of the live C. After blood collection, it was temporarily stored at 4°C.

[0044] 2. Peripheral blood cell culture

[0045] Cell culture operations were performed under sterile conditions using a modified commercially available Eagle basal medium. The medium modified formula was added on the basal medium, 20mM 4-hydroxyethylpiperazineethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acids, 1mM sodium pyruvate, 1mM penicillin and strepto Mycin double antibody, 55μM β-mercaptoethanol, 15% fetal bovine serum, 5ng / mL recombinant human basic fibroblast growth factor. Add 200 μL of whole blood per 3 ml of medium, and add 750 μg of PHA-P.

[0046] 3. Colchicine treatment

[0047] Blood cells were cultured at 28°C (CO 2 The ...

Embodiment 3

[0051] 1. Peripheral blood sampling

[0052] Using a commercially available negative pressure blood collection tube (containing sodium heparin) and a sterilized needle, collect 1 ml of peripheral blood from the cervical sinusoids of the live C. After blood collection, it was temporarily stored at 4°C.

[0053] 2. Peripheral blood cell culture

[0054] Cell culture operations were performed under sterile conditions using a modified commercially available Eagle basal medium. The medium modified formula was added on the basal medium, 20mM 4-hydroxyethylpiperazineethanesulfonic acid, 1mM glutamate, 2nM sodium selenite, 1mM non-essential amino acids, 1mM sodium pyruvate, 1mM penicillin and strepto Mycin double antibody, 55μM β-mercaptoethanol, 15% fetal bovine serum, 5ng / mL recombinant human basic fibroblast growth factor. Add 200 μL of whole blood per 3 ml of medium, and add 750 μg of PHA-P.

[0055] 3. Colchicine treatment

[0056] Blood cells were cultured at 28°C (CO 2 Th...

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Abstract

The invention discloses a method for preparing a pelochelys bibroni chromosome specimen by using peripheral blood cells, and belongs to the field of cytogenetics. Peripheral blood of a pelochelys bibroni is collected, and a pelochelys bibroni chromosome metaphase specimen which is convenient to observe and clear in image is prepared through the steps of blood cell culture, colchicine treatment, staining and slide preparation and the like. According to the method of the invention, the problem of large damage to animals in an existing aquatic animal chromosome specimen preparation method is solved, non-damage sampling is realized, only a small amount of peripheral blood of the sample needs to be collected, and the influence on the living state of the animals is small. The slide preparation method is simple and rapid; the metaphase of the pelochelys bibroni chromosome can be obtained 72 hours after blood sampling, observation is facilitated, and a beneficial tool is provided for further developing pelochelys bibroni genome and evolution related research and realizing more sufficient protection of the pelochelys bibroni .

Description

technical field [0001] The invention relates to the field of cytogenetics, in particular to a method for using peripheral blood cells to prepare electrochromosomal specimens. Background technique [0002] Chromosome is an important component of the nucleus and the main material carrier of biological genetic information. The karyotype of organisms not only has the characteristics of species, but also reflects the history of biological evolution. Chromosomal karyotype analysis is an important research content in aquatic genetic breeding and germplasm identification. [0003] At present, there are three conventional methods for preparing chromosomes in aquatic animals. 1) In vivo cell culture method, that is, injecting phytohemagglutinin (PHA-P) into the abdominal cavity or chest cavity to stimulate B in hematopoietic organs such as the head kidney or spleen of experimental animals. A large number of lymphocytes proliferate, and then treated with colchicine, so that the cell d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N1/28A61B5/153
CPCA61B5/15003A61B5/153A61B2503/40A61B2503/42G01N1/28G01N1/30G01N2001/302G01N2001/305
Inventor 陈辰洪孝友朱新平刘晓莉曹建萌仇全波
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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