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Forward genetic screen using transgenic calcium reporter aequorin to identify novel targets in calcium signaling
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View Archive Info| Field | Value | |
| Title |
Forward genetic screen using transgenic calcium reporter aequorin to identify novel targets in calcium signaling
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| Creator |
Mittal, Deepika
Mishra, Shruti Prajapati, Ramgopal Vadassery, Jyothilakshmi |
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| Subject |
Aequorin
Arabidopsis Calcium signaling EMS Forward genetic screen |
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| Description |
Accepted date: 1 June 2020
Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets. The video component of this article can be found at https://www.jove.com/video/61259/ We thank National Institute of Plant Genome Research - Phytotron Facility for plant growth, Bombay Locale for the video shoot, and the Department of Biotechnology- eLibrary Consortium for providing access to e-resources. This work was supported by the Department of Biotechnology, India through the National Institute of Plant Genome Research Core Grant, Max Planck Gesellschaft-India Partner Group program; and CSIR-Junior Research Fellowship (to D.M and S.M) and Department of Biotechnology-Junior Research Fellowship (to R.P). https://www.jove.com/v/61259/forward-genetic-screen-using-transgenic-calcium-reporter-aequorin-to |
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| Date |
2020-06-11T08:30:07Z
2020-06-11T08:30:07Z 2020 |
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| Type |
Article
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| Identifier |
Jove-Journal of Visualized Experiments, 162: e61259
1940-087X doi:10.3791/61259 https://www.jove.com/video/61259/forward-genetic-screen-using-transgenic-calcium-reporter-aequorin-to http://223.31.159.10:8080/jspui/handle/123456789/1068 https://www.jove.com/v/61259/forward-genetic-screen-using-transgenic-calcium-reporter-aequorin-to |
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| Language |
en_US
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| Format |
application/pdf
application/octet-stream |
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| Publisher |
Journal of Visualized Experiment
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