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Ein schnelles Protokoll zur Integration extrachromosomaler Arrays mit hoher Übertragungsrate in d...
Ein schnelles Protokoll zur Integration extrachromosomaler Arrays mit hoher Übertragungsrate in d...
JoVE Journal
Biology
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JoVE Journal Biology
A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Ein schnelles Protokoll zur Integration extrachromosomaler Arrays mit hoher Übertragungsrate in das Genom von C. elegans

Full Text
9,546 Views
06:33 min
December 9, 2013

DOI: 10.3791/50773-v

Marie-Christine Mariol1,2, Ludivine Walter1,2, Stéphanie Bellemin1,2, Kathrin Gieseler1,2

1Université Claude Bernard Lyon, 2Centre de Génétique et de Physiologie Moléculaires et Cellulaires,CNRS UMR 5534

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Overview

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultraviolet (UV) irradiation. This method is particularly effective for transgenic lines that transmit extrachromosomal arrays at a high rate.

Key Study Components

Area of Science

  • Neuroscience
  • Genetics
  • Transgenics

Background

  • Transgenic organisms are vital for studying gene function.
  • Extrachromosomal arrays can facilitate gene expression.
  • UV irradiation is a method for integrating genetic material.
  • High transmission rates are crucial for successful experiments.

Purpose of Study

  • To develop a protocol for integrating transgenic arrays into C. elegans.
  • To enhance the efficiency of transgene transmission.
  • To provide a low material consuming method for researchers.

Methods Used

  • Select transgenic lines with high transmission rates (ideally >80%).
  • UV irradiate selected L4 larvae.
  • Screen F2 plates for fluorescent transgenic worms.
  • Confirm inheritance by selecting fluorescent F3 generation animals.

Main Results

  • Successful integration of transgenic arrays into the genome.
  • High rates of transgene inheritance observed.
  • Protocol demonstrated efficiency and low material usage.
  • Fluorescent screening enabled easy identification of transgenic lines.

Conclusions

  • The protocol is effective for creating stable transgenic lines.
  • UV irradiation is a viable method for genetic integration.
  • This approach can be adapted for various transgenic applications.

Frequently Asked Questions

What is the main advantage of this protocol?
It is rapid and consumes low amounts of material while achieving high transgene transmission rates.
Can this method be used for other organisms?
This protocol is specifically designed for Caenorhabditis elegans.
What is the significance of using UV irradiation?
UV irradiation facilitates the integration of extrachromosomal arrays into the genome.
How can researchers confirm successful integration?
By screening for fluorescent transgenic worms in the F3 generation.
Is there a specific age for larvae to be used?
L4 larvae are recommended for UV irradiation in this protocol.
What is the expected transmission rate for optimal results?
A transmission rate greater than 80% is ideal for this method.

Dieses Protokoll beschreibt ein schnelles und materialsparendes Verfahren zur Integration von transgenen extrachromosomalen Arrays in das Genom von Caenorhabditis elegans mittels ultravioletter (UV) Bestrahlung. Darüber hinaus eignet sich dieses Protokoll besonders gut für transgene Linien, die extrachromosomale Arrays mit hoher Geschwindigkeit übertragen.

Das übergeordnete Ziel dieses Verfahrens ist es, transgene extrachromosomale Arrays mittels ultravioletter Bestrahlung in das Serratus-Elegance-Genom zu integrieren. Zuerst wählen Sie die transgene Linie mit der höchstmöglichen Transmissionsrate, idealerweise mehr als 80 %, dann UV-Bestrahlung der ausgewählten transgenen L vier Larven und wählen Sie vier der fluoreszierenden transgenen F zwei Tiere aus jeder ausgewählten F1-Platte aus. Nächster Bildschirm, die F zwei Platten für 100% fluoreszierend transgen.

F drei Würmer. Letztendlich kann die Auswahl eines fluoreszierenden Tieres der dritten Generation die 100%ige Vererbung des Transgens zeigen. Dieser experimentelle Ansatz eignet sich gut für transgene Linien, die zusätzliche Chromosomenarrays mit hoher Geschwindigkeit übertragen.

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Schlüsselwörter: C. elegans Extrachromosomale Arrays transgene Linien UV-Bestrahlung Genomintegration hohe Übertragungsrate Mendelsche Segregation nichtmosaische Expression Rapid Protocol Transgene Expression

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