Reagent kit and method for dissociating animal embryo

Abstract

The invention discloses a reagent kit and method for dissociating an animal embryo. The reagent kit for dissociating an animal embryo disclosed by the invention comprises reagents of which the names are respectively an enzymolysis solution C, an enzymolysis solution D and an enzymolysis solution E, wherein the enzymolysis solution C consists of cytochalasin storing fluid and TrypLE digestive fluid, and the volume ratio of the cytochalasin storing fluid to the TrypLE digestive fluid is (3 to 47) to (3 to:97); the enzymolysis solution D is fluid consisting of fetal calf serums, a pronase solution and a TCM-HEPES buffer solution, and the volume ratio of the fetal calf serums to the pronase solution to the TCM-HEPES buffer solution is (1 to 1 to 1) to (1 to 2 to 1); and the enzymolysis solution E consists of cytochalasin storing fluid and Accutase cell separation fluid, and the volume ratio of the cytochalasin storing fluid to the Accutase cell separation fluid is (3 to 47) to (3 to 97). Experiment proves that when the reagent kit and method disclosed by the invention are used for dissociating the animal embryo, the dissociating efficiency is high, the embryo of an animal of species, which cannot be dissociated by an existing method, can be dissociated, the reagent kit and the method are suitable for the embryos at different developmental stages, and hurt to cells in the dissociating process can also be reduced.

Description

technical field [0001] The invention relates to a kit and a method for dissociation of animal embryos in the field of biotechnology. Background technique [0002] Now the most common method for dissociation of tissue or cultured cells into single cells in laboratories is enzymatic hydrolysis, and trypsin is the most commonly used enzyme for dissociation of tissues and cells in laboratories because of its cheap price and strong effect. However, enzymatic hydrolysis with trypsin has the following disadvantages: 1) Due to the particularity of the embryo (the outer layer has a zona pellucida, the cells are densely connected, and there are different developmental stages such as two-cell, four-cell to blastocyst stage), trypsin is used Enzymatic hydrolysis has low efficiency and cannot be dissociated for some species of embryos such as pig embryos; 2) The dissociation efficiency is not high. In order to meet the research requirements, more embryos need to be dissociated, which inc...

AI Technical Summary

Problems solved by technology

However, enzymatic hydrolysis with trypsin has the following disadvantages: 1) Due to the particularity of the embryo (the outer layer has a zona pellucida, the cells are densely connected, and there are different developmental stages such as two-cell, four-cell to blastocyst stage), trypsin Enzymatic hydrolysis has low efficiency and cannot be dissociated for some species of embryos such as pig embryos; 2) The dissociation efficiency is not high. In order to meet the research requirements, more embryos need to be dissociated, which increases the cost of the experiment; 3) Embryos of different developmental stages Different methods or enzyme concentrations need to be used, which may lead to inhomogeneity of results due to different methods; 4) Trypsin itself has a relatively strong effect, which is easy to cause cell membrane damage and cell apoptosis, which is not conducive to follow-up research, etc.

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