Chapter title |
Generating and Identifying Axolotls with Targeted Mutations Using Cas9 RNA-Guided Nuclease.
|
---|---|
Chapter number | 22 |
Book title |
Salamanders in Regeneration Research
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2495-0_22 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2494-3, 978-1-4939-2495-0
|
Authors |
G Parker Flowers, Craig M Crews, G. Parker Flowers, Craig M. Crews, Flowers, G. Parker, Crews, Craig M. |
Abstract |
The CRISPR/Cas9 RNA-guided nuclease now enables a reverse genetics approach to investigate the function of genes of interest during regeneration in the axolotl. The process of generating the constructs necessary for targeting a gene of interest is considerably less labor intensive than for other methods of targeted mutagenesis such as Zinc finger nucleases or Transcription activator-like effector nucleases. Here, we describe the identification of targetable sequences in the gene of interest, the construction of unique guide RNAs, the microinjection of these RNAs with Cas9-encoding mRNA, the selection of well-injected animals, and an inexpensive, PCR-based method for identifying highly mutagenized animals. |
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Unknown | 1 | 100% |
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Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 19 | 100% |
Demographic breakdown
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Student > Ph. D. Student | 7 | 37% |
Researcher | 5 | 26% |
Student > Bachelor | 1 | 5% |
Professor | 1 | 5% |
Other | 1 | 5% |
Other | 0 | 0% |
Unknown | 4 | 21% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 5 | 26% |
Agricultural and Biological Sciences | 5 | 26% |
Chemistry | 1 | 5% |
Medicine and Dentistry | 1 | 5% |
Unknown | 7 | 37% |