embryo segmentation

Summary

Embryo splitting refers to the technique of splitting an animal's pre-implantation embryo into two, four or even eight embryos using a micromanipulator. Embryo splitting is an important means to study cell differentiation, early embryonic development and embryonic cell totipotency, and has wide application in developmental biology, animal physiology and production practice. By dividing embryos, the number of homogeneous embryos can be increased, and the production of livestock and the promotion of good breeds can be improved. Embryo segmentation technology also provides a method and means to obtain ovoid spheres for cell nuclear transfer, embryo sex identification, embryo chimerism and so on.

Principle

The principle of embryo segmentation is that mammalian early embryos have different degrees of developmental adjustment ability, i.e., after removing or destroying part of the embryo, the remaining part continues to become a complete individual by adjusting its developmental direction; whereas, when two early embryos are artificially fused together, the two are only able to develop into a complete embryonic body through the mechanism of intercellular remodeling.

This indicates that the developmental process of mammalian early embryos is adjustable, and the earlier the adjustment, the greater the adjustment, but with the development of the embryo, the developmental direction of the cells is slowly fixed, until the final adjustment ability is completely lost.

Operation method

embryo segmentation

Principle

The principle of embryo segmentation is that mammalian early embryos have different degrees of developmental adjustment ability, i.e., after removing or destroying part of the embryo, the remaining part continues to become a complete individual by adjusting its developmental direction; whereas, when two early embryos are artificially fused together, the two are only able to develop into a complete embryonic body through the mechanism of intercellular remodeling. This indicates that the developmental process of mammalian early embryos is adjustable, and the earlier the adjustment, the greater the adjustment, but with the development of the embryo, the developmental direction of the cells is slowly fixed, until the final adjustment ability is completely lost.

Materials and Instruments

Equipment:
① Stereomicroscope,
② inverted microscope or micro-manipulator
③ egg-holding needle, dividing needle
④Microtome
⑤Cell culture incubator
Reagents:
①Materials: Animals
②75% ethanol
②75% ethanol ③0.2%～0.5% streptavidin
④Culture medium with bovine serum albumin added.
⑤Agar

Move

The basic process of embryo splitting can be divided into the following steps:

(I) Preparation of embryo splitting apparatus

(1) Micro-segmentation: before embryo segmentation, it is necessary to make egg-holding needles and segmentation needles, the egg-holding needles are required to have bluntly rounded ends, with outer diameters similar to the diameters of the fixed embryos, and inner diameters of 20-30 μm in general.

(2) Currently, there are two kinds of splitting needles: glass needle and microtome; the glass needle is generally made of solid glass rod, and its tip is required to be bent into a sickle shape; the microtome is made of a sharp metal blade glued together with a thin glass rod.

(B) pretreatment of embryos

Embryos are treated with 0.2% to 0.5% streptavidin for a short time before splitting, and after the zona pellucida is softened and thinned, the embryos are transferred to the culture medium with bovine serum albumin.

(iii) Splitting of embryos

Place the treated embryos together with a small amount of culture medium on a disposable petri dish. Under the micromanipulator, install the egg-holding needle on the left operating arm and the dividing needle on the right operating arm, and control the left and right operating arms through the operating table.

Adjust the coarse adjustment of the left operating arm to make it close to the embryo and at the optimal operating angle, and then adjust the negative pressure of the microinjector to make the egg-holding needle attract and hold the embryo to be divided.

On the other side of the segmentation needle is adjusted to the upper part of the embryo and the segmentation needle is slowly lowered, and then it is moved downward from the equidistant line of the embryo, and the embryo is gently pressed down, and when the needle touches the bottom of the Petri dish, it can be moved back and forth a little bit to split the embryo into two.

(D) Cultivation of split embryos

In order to improve the pregnancy rate and utilization rate of cut embryo transfer, the split embryos can be put into empty zona pellucida, or embedded in agar and transferred into intermediate recipients for in vivo culture or direct in vitro culture.

(E) Preservation and transfer of split embryos

(1) Frozen embryos are stored in liquid nitrogen at -196 ℃. In order to improve the transferability of frozen embryos, the split embryos should be cultured in vivo or in vitro until they reach the stage of mulberry embryo or blastocyst before freezing.

(2) For embryo transfer, segmented embryos can be transferred to different parts of the recipient's fallopian tube when they have developed to 4-8 cells, and to different parts of the recipient's uterus when they have developed to the 16-cell cleavage sphere or mulberry embryo.

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