NDLI: Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE)

Content Provider	Springer Nature Link
Author	Cooper, Caitlin A. Challagulla, Arjun Jenkins, Kristie A. Wise, Terry G. O’Neil, Terri E. Morris, Kirsten R. Tizard, Mark L. Doran, Timothy J.
Copyright Year	2016
Abstract	Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.
Starting Page	331
Ending Page	347
Page Count	17
File Format	PDF
ISSN	09628819
Journal	Transgenic Research
Volume Number	26
Issue Number	3
e-ISSN	15739368
Language	English
Publisher	Springer International Publishing
Publisher Date	2016-11-28
Publisher Place	Cham
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Gene editing CRISPR/Cas9 Avian models Genome engineering Animal Genetics and Genomics Plant Genetics & Genomics Transgenics Biomedical Engineering/Biotechnology Genetic Engineering Molecular Medicine
Content Type	Text
Resource Type	Article
Subject	Genetics Animal Science and Zoology Biotechnology Agronomy and Crop Science

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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