Method for chicken embryo cultivation and in vivo electroporation of gene

Abstract

The present invention discloses a method for chicken embryo cultivation and in vivo electroporation of gene. The method comprises the following steps: carrying out shelled culture: a treatment of in situ electroporation of gene is performed for the chicken embryo with the shell after culturing for 2-3 days; carrying out shell removing culture: a fertilized egg is cultured for 3 days, the shell is removed from the fertilized egg, then the fertilized egg without the shell is placed in a culture container, the culture container is placed in a constant temperature and constant humidity incubator to carry out culture; carrying out a treatment step of in situ electroporation of gene: after carrying out shelled culture for 2-3 days, 0.5-1 mul of plasmid (0.5 mug / mul) is injected to spinal cord by using a microinjection capillary glass needle through a stereomicroscope, or after carrying out shell removing culture for 5-6 days, 0.5-1 mul of plasmid (0.5 mug / mul) is injected to an optic tectum position by using the microinjection capillary glass needle through the stereomicroscope, then electrodes are placed on both sides of the spinal cord injected with the plasmid or the optic tectum position injected with the plasmid to carry out the electroporation, wherein the positive electrode is placed on the side having the gene requiring the electroporation; then carrying out tissue sampling, embedding and slicing. According to the present invention, with optimizing the traditional culture method, the shelled chicken embryo culture technology and the shell removing chicken embryo culture technology are established, the survival rate of the chicken embryo culture is improved, and the basis is provided for the chick embryo in vivo in situ electroporation of gene.

Description

technical field [0001] The invention relates to the technical field of developmental biology and transgene, in particular to the technical field of chicken embryo culture and in situ electrical transgene of chicken embryo. [0002] Background technique [0003] Chicken embryo is an important experimental animal model in the research fields of tissue embryo development and molecular biology. Compared with other experimental animals, chicken embryo has many advantages such as short development cycle, clear genetic and physiological background, simple and easy to obtain, and easy to operate. In addition, chicken embryos can be manipulated under a stereo microscope, and their subsequent development can also be monitored and videotaped in vitro, which is an advantage that embryos of other species cannot replace. With the publication of the chicken genome sequence and the development of gene function gain and loss research techniques in chicken eggs, the chicken embryo model has ...

AI Technical Summary

Problems solved by technology

The advantage of in-shell culture is that the operation is simple, the survival rate of culture is high, and the survival time of embryos in the eggshell environment is long, which is suitable for the development of early gene transfection technology. Dyeing is inconvenient. After 5 days of embryonic development, it is difficult to achieve injection and electric shock. Therefore, it can only be used for transfection of chicken embryos cultured within 5 days. In the later stage of embryonic development, the model cultured with shell It is not suitable for the study of related gene functions, because when the eggshell is removed, blood vessels and eggshells will adhere to each other when the eggshell is removed, and there is no guarantee that part of the eggshell will be removed without destroying the blood vessels.

The advantage of shelled culture is that it solves the shortcomings of shelled culture, and can realize the development of gene transfection technology for larger embryos after 5 days. In addition, during the culture process, the embryos are always floating above the egg white, which is conducive to injection and injection. Observation and other processes, Yalcin HC et al. also proved that the dehulling culture of chicken embryos is conducive to the development of imaging and microsurgery (Yalcin HC, Shekhar A, Rane AA, Butcher JT.J Vis Exp, 23 (44): 10.3791 / 2154 , 2010), the disadvantages are that the cultivation operation is difficult, easy to be polluted, and the survival rate of culture is low. It is necessary to ensure the integrity of the vitelline membrane and the blood vessels during the process of removing the eggshell. The yolk plays a very important role in the development of chicken embryos. function, the rupture of the vitelline membrane or the loss of the yolk will affect the development of the embryo

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