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A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry
Published on: July 12, 2017
Isolation of teleost primordial germ cells using flow cytometry
Rie Goto-Kazeto1, Taiju Saito, Misae Takagi
1Nanae Fresh Water laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Hakodate, Hokkaido, Japan. goto-kazeto@hokudai-nanae.jp
The International Journal of Developmental Biology
|February 9, 2011
Summary
Primordial germ cells (PGCs) can be isolated from fish embryos using GFP labeling and flow cytometry without creating transgenic fish. Isolated PGCs retain their ability to migrate, offering a valuable tool for germ cell research.
Area of Science:
- Developmental Biology
- Reproductive Biology
- Genetics
Background:
- Primordial germ cells (PGCs) are crucial for transmitting genetic information across generations.
- Previous studies utilized green fluorescent protein (GFP) mRNA constructs to label PGCs in various fish species.
Purpose of the Study:
- To isolate teleost PGCs using flow cytometry (FCM) and GFP labeling.
- To investigate germ cell migration to the gonadal region using isolated PGCs.
Main Methods:
- Injection of a GFP-nos1 3UTR mRNA construct into early-stage embryos of zebrafish, medaka, and goldfish.
- Enzymatic disaggregation of embryos and isolation of GFP-positive cells via FCM.
- In situ hybridization to confirm PGC identity (vasa RNA expression) and in vivo transplantation to assess migration.
Main Results:
- PGCs were successfully labeled and isolated from zebrafish, medaka, and goldfish using FCM.
- Sorted zebrafish PGCs expressed vasa RNA, confirming their identity.
- Approximately 30% of sorted zebrafish PGCs migrated to the gonadal region in host embryos.
Conclusions:
- This method allows for the isolation of PGCs from fish without the need for transgenic organisms.
- Isolated PGCs maintain their migratory capabilities.
- The technique is valuable for studying PGCs across diverse fish species.
