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Cryopreservation of Xenopus transgenic lines
Daniel R Buchholz1, Liezhen Fu, Yun-Bo Shi
1Unit on Molecular Morphogenesis, Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.
Molecular Reproduction and Development
|December 4, 2003
Summary
Cryopreserved sperm from Xenopus laevis (African clawed frogs) can regenerate transgenic lines. This method offers an inexpensive way to preserve and distribute valuable transgenic frog models for research.
Area of Science:
- Developmental Biology
- Genetics
- Reproductive Biology
Background:
- Xenopus laevis is a key model organism for molecular, cellular, and developmental studies.
- Sperm-mediated transgenesis allows gene function studies in vertebrate development.
- Maintaining transgenic lines is costly and labor-intensive.
Purpose of the Study:
- To investigate sperm cryopreservation for preserving transgenic Xenopus laevis lines.
- To establish a cost-effective method for maintaining and distributing transgenic frog models.
Main Methods:
- Cryopreservation of Xenopus laevis sperm.
- In vitro fertilization (IVF) attempts with cryopreserved sperm.
- Microinjection of cryopreserved sperm nuclei into oocytes.
- Generation of a transgenic line with a double promoter construct (gamma-crystallin and heat shock-inducible promoters) driving GFP and ST3-GFP expression.
- Analysis of F1 animal phenotype following heat-shock induction.
Main Results:
- Cryopreserved sperm were viable but not fertile under standard IVF conditions.
- Successful regeneration of a transgenic Xenopus line by microinjecting cryopreserved sperm nuclei.
- Demonstrated functional transmission of the ST3-GFP transgene in F1 offspring after heat-shock induction.
Conclusions:
- Sperm cryopreservation followed by nuclear microinjection is an effective method for preserving transgenic Xenopus laevis lines.
- This technique provides an inexpensive and convenient approach for maintaining and distributing transgenic frog models.
- Simplified nuclear microinjection facilitates wider use of transgenic Xenopus laevis in functional studies.