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Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos
Published on: March 10, 2016
Screening for transgenic Japanese quail offspring.
Greg Poynter1, David Huss, Rusty Lansford
1Division of Biology and Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.
Cold Spring Harbor Protocols
|February 12, 2010
Summary
This study presents a method for isolating genomic DNA from Japanese quail chorioallantoic membranes (CAM) to screen for transgenes. This technique reduces hatchling stress and simplifies genetic analysis for the G1 generation.
Area of Science:
- Animal Genetics
- Molecular Biology
- Developmental Biology
Background:
- Screening Japanese quail hatchlings for germ-line transmission of transgenes is crucial for genetic studies.
- Traditional methods like blood draws can cause stress and require specialized handling.
- Efficient and non-invasive DNA isolation methods are needed for large-scale genetic screening.
Purpose of the Study:
- To describe a protocol for isolating high-quality genomic DNA from the chorioallantoic membrane (CAM) of Japanese quail hatchlings.
- To present a polymerase chain reaction (PCR) genotyping method for screening transgenes in the isolated genomic DNA.
- To offer an alternative to blood-based DNA collection, minimizing stress on young birds.
Main Methods:
- Isolation of genomic DNA from the chorioallantoic membrane (CAM) remaining within the hatched egg.
- Quantification of isolated genomic DNA yield (≥50 microg per CAM).
- Application of polymerase chain reaction (PCR) for transgene screening of the genomic DNA samples.
Main Results:
- Successful isolation of high-quality genomic DNA from quail CAM, yielding 50 microg or more per sample.
- Demonstration of PCR genotyping as an effective method for detecting transgenes in the isolated DNA.
- Validation of the CAM DNA isolation method as a less stressful alternative to blood collection.
Conclusions:
- The described CAM DNA isolation protocol provides a reliable and efficient method for genetic screening of Japanese quail hatchlings.
- PCR genotyping of CAM DNA is suitable for screening both nonfluorescent and certain fluorescent transgenes.
- This method significantly reduces hatchling stress and simplifies the process of identifying G1 generation transgenic birds.
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