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Method for separating and purifying oogonia of paralichthys olivaceus

An oogonia, separation and purification technology, applied in cell dissociation methods, biochemical equipment and methods, reproductive cells, etc., to achieve the effect of little loss of quantity and easy operation

Active Publication Date: 2018-07-10
中国水产科学研究院北戴河中心实验站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, oogonium transplantation in flounder has not been reported

Method used

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  • Method for separating and purifying oogonia of paralichthys olivaceus
  • Method for separating and purifying oogonia of paralichthys olivaceus
  • Method for separating and purifying oogonia of paralichthys olivaceus

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Embodiment

[0043] This embodiment provides a method for separating and purifying flounder oogonia.

[0044] ①Experimental fish

[0045] Select 4 18-month-old female flounder fish, measure the total length and weight, and then cut off the spine to bleed. After the blood dries up, collect the ovarian tissue and weigh it, wash it with PBS (pH 7.2) once, and cut a 0.5cm 3 Left and right gonad tissues were fixed with Born's test solution. After fixing for 24 hours, replace it with 70% alcohol and save it for later use. See Table 1 for the growth statistics of the 18-month-old flounder.

[0046] Table 1 The growth of 18-month-old flounder

[0047]

[0048] ② Design and production of flounder Vasa antibody (αVasa)

[0049] The flounder Vasa protein sequence (GenBank sequence number: XP_019957862.1) 1-204aa was used as the antigen sequence to construct a plasmid and express the purified protein to prepare the antigen for the preparation of the flounder Vasa rabbit polyclonal antibody (pro...

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Abstract

The invention relates to a method for separating and purifying oogonia of paralichthys olivaceus. The method comprises the following steps of 1), stripping tunica albuginea from the gonadal tissue offemale paralichthys olivaceus, cutting up, centrifuging, then digesting an obtained deposit by using combined enzyme liquid, filtering obtained cell digestive juice through a cell screen, centrifuging, and then resuspending to obtain cell suspension, wherein the combined enzyme liquid is prepared from trypsin and a DNA (Deoxyribonucleic Acid) enzyme I; 2), preparing Percoll liquid under physiological osmotic pressure to different concentration gradients, lightly flowing each level of well prepared Percoll gradient liquid into a centrifugal tube along a tube wall layer by layer from a high gradient to a low gradient to prepare for forming Percoll gradient liquid, and putting the cell suspension on the uppermost layer of the gradient; centrifuging, and then absorbing two uppermost layers ofcell bands to obtain the oogonia of the paralichthys olivaceus. The method provided by the invention is easy to operate; the whole operation is controllable; the oogonia of the paralichthys olivaceuscan be successfully separated out by adopting the method; the proportion of the oogonia is 80 percents or above, and the quantitative loss of the oogonia before and after purification is low.

Description

technical field [0001] The invention relates to the fields of reproductive biology and biotechnology, in particular to a method for separating and purifying flounder oocytes. Background technique [0002] In most species, primordial germ cells, spermatogonia, and oogonia have been shown to be stem cells that self-renew and produce functional gametes. Among them, in invertebrates and vertebrates with a mass egg-laying reproductive strategy, oogonia are progenitor cells present in the ovary of female animals, which perform the function of stem cells and ensure the continuous production of eggs throughout the animal's reproductive age Mother cells, which eventually form ova. [0003] In recent years, germ cell transplantation technology (Germ cell transplantation, GCT) has been widely used in aquaculture and endangered species protection, both of which have obtained functional gametes or offspring derived from donors. Generally, primordial germ cells (PGCs), spermatogonia (Sp...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2509/00
Inventor 任玉芹孙朝徽王玉芬于清海周勤宋立民姜秀凤王青林司飞
Owner 中国水产科学研究院北戴河中心实验站
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