This protocol is an excerpt from&#160;Mariol et al,&#160;<a href="Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome">A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the&#160;C. elegans&#160;Genome</a>,&#160;J. Vis. Exp.&#160;(2013).
1. UV Irradiation and Recovery of Transgenic Worms
<ol>
	<li>Worm irradiation:
	<ol>
		<li>Pick 30-100 fluorescent transgenic L4 animals onto separate culture plates (15-20 animals by plate).</li>
		<li>Place the plates, with the lids removed, in a UV cross-linker. Irradiate the worms at 0.012 J/cm2. Note that strains are more or less sensitive to UV and that results can be variable depending on the UV irradiator used. A dose range between 0.010-0.015 J/cm2&#160;can be tested.</li>
	</ol>
	</li>
	<li>Worm recovery after irradiation:
	<ol>
		<li>Place plates with irradiated worms overnight at 15 &#176;C for recovery.</li>
		<li>Check for the number animals that are alive. In our hands, a survival rate of around 80-90% is adapted for an efficient irradiation.</li>
		<li>Grow the irradiated animals at 15-25 &#176;C until the progeny has reached the developmental stage allowing for the observation of the coinjection marker expression.</li>
	</ol>
	</li>
</ol>
2. Isolation of Integrated Transgenic Lines
<ol>
	<li>Selection of F1 animals:
	<ol>
		<li>Single 150-200 fluorescent F1 animals onto separate culture plates. For highly transmitting lines (&#8805;80%), 100 F1 animals are enough.</li>
		<li>Keep these F1 plates at 15-25 &#176;C until the progeny reaches an appropriate stage for the observation of fluorescent F2 animals.</li>
		<li>Discard all F1 plates exhibiting either&#160;i)&#160;no progeny, indicating that the F1 animal was sterile or died, or&#160;ii)&#160;no or only few fluorescent F2 animals, indicating that the F1 animal did not transmit the transgene at the expected rate. This step is quick and represents a real advantage when compared to the standard protocol that requires an estimation of the percentage of fluorescent F2 worms in order to select F1 plates exhibiting &#8805;75% fluorescent progeny (Table 1).</li>
	</ol>
	</li>
	<li>Selection of F2 animals:
	<ol>
		<li>Single four fluorescent transgenic F2 animals from each selected F1 plate. When using fluorescent transgenes (for example mCherry, gfp, or dsRed), pick F2 worms with a high level of fluorescence as this could indicate that these animals are homozygous for the integrated array. Note that when picking F2 animals it is critical not to carry along eggs or larvae, in order to avoid false negatives in the next step.</li>
		<li>Grow F2 animals at 15-25 &#176;C.</li>
	</ol>
	</li>
	<li>Isolation and validation of integrated transgenic strains:
	<ol>
		<li>Screen F2 plates for 100% fluorescent transgenic F3 worms. The screen is quite rapid as the presence of a single nonfluorescent worm indicates that the plate should be thrown away. A F2 animal homozygous for the integrated transgene will give 100% homozygous fluorescent progeny.</li>
		<li>Single eight fluorescent F3 animals from selected plates to confirm the 100% inheritance of the transgene. In theory, picking one animal is enough at this step. However, picking eight fluorescent F3 animals may be necessary to avoid selecting unfertile or unhealthy animals as irradiation can randomly induce mutations that affect genes important for worm physiology.</li>
		<li>If possible keep several independent integrated transgenic strains,&#160;i.e.&#160;strains recovered from different F1 animals.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>U.V. cross-linker</td><td>Fischer Scientific</td><td></td><td>Wavelength 254 nm</td></tr><tr><td>Microscope SteREO Discovery V8</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Petri dishes, 60 mm</td><td>CML</td><td>BPS55E6</td><td></td></tr><tr><td>Platinium wire 0.25 mm</td><td>Alfa Aesar</td><td>6/4/7440</td><td>To make a pick for worms&amp;nbsp;</td></tr></tbody></table>

uv irradiation: a method to integrate extrachromosomal arrays

Source: Mariol, M. C et al. <a href="Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome">A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the&#160;C. elegans&#160;Genome.</a>&#160;J. Vis. Exp.&#160;(2013).
Microinjection of transgenes usually results in C. elegans strains with large extrachromosomal arrays, which result in variable expression and transmission of the transgene.&#160; UV Irradiation can induce chromosomal integration of the array and more stable transgene transmission.

<img alt="Table 1" src="/files/ftp_upload/20169/20169tbl1.jpg" /> 
Table 1. Comparison of the standard and improved protocols for extrachromosomal arrays integration into the&#160;C. elegans&#160;genome.&#160;Two nonintegrated transgenes transmitting at about the same frequency were integrated using the standard or the improved protocol. The main difference lies in the fact that in the standard protocol a screen of the proge...

UV Irradiation: A Method to Integrate Extrachromosomal Arrays

Source: Mariol, M. C et al. A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the&#160;C. ...

- Creating extrachromosomal DNA arrays by microinjection-- the simplest method to generate transgenic worm strains-- results in genetic elements that are not stably transmitted during cell division and, therefore, exhibit variable inheritance rates and expression levels. The goal of integrating the array into a chromosome is to lessen this genetic instability and variability. To do so, start with an age-synchronized population of L4 transgenic worms, which carry an extrachromosomal array previously introduced by microinjection and allow them to grow into gravid adults. Transfer these adults to new plates and allow them to lay eggs until there are at least 30 eggs on each plate. Remove the adults and allow the eggs to grow up until they reach the L4 stage. Then, irradiate the worms with UV light. This causes random double-strand breaks in the genomic DNA where the worms DNA repair machinery may arbitrarily incorporate the array. After an overnight recovery period, count the number of surviving worms. A small percentage of dead worms indicates efficient irradiation. Observe subsequent generations to isolate homozygous integrated transgenic lines. In the example protocol, we will integrate a high transmission rate extrachromosomal array carrying a fluorescent co-injection marker expressed in the pharynx. - Evaluate the transmission rate of the transgenic line to be integrated. For each transgenic line, pick 10 fluorescent gravid adults onto 10 separate culture plates under a fluorescent stereoscope. Culture the animals in a worm incubator set at a temperature permissive to reproduction of the genetic background. Monitor the progeny each day to evaluate which developmental stage the co-injection marker is expressed and can be best observed. Using fluorescent stereoscopy, evaluate the transmission rate of the progeny by determining the percentage of fluorescent progeny. For the transgene integration, select the transgenic line with the highest transmission rate. Obtain a population of transgenic animals synchronized at the L4 larval stage for integration. Pick 30 fluorescent gravid adults onto five culture plates. After the worms have laid eggs for three to four hours at 15 degrees Celsius, check for the presence of at least 30 eggs by plate, and then, eliminate the adults from the plates. Culture the animals in a worm incubator until the progeny reach the L4 larval stage. Place each plate containing 15 to 20 fluorescent transgenic L4 animals in a UV crosslinker with the lids removed. Irradiate the worms. For recovery, incubate the irradiated worms overnight at 15 degrees Celsius. Check for the number of animals that are alive. In our hands, a survival rate of around 80% to 90% is adaptive for an efficient irradiation. Grow the irradiated animals at 15 to 25 degrees Celsius until the progeny have reached the developmental stage allowing for the observation of the co-injection marker expression.

uv-irradiation-a-method-to-integrate-extrachromosomal-arrays

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JoVE Encyclopedia of Experiments

Biology

Caenorhabditis elegans (worm)

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紫外线照射:一种整合染色体外阵列的方法

Microinjection of transgenes usually results in C. elegans strains with large extrachromosomal arrays, which result in variable expression and transmission of the transgene.&#160; UV Irradiation can induce chromosomal integration of the array and more stable transgene transmission.

Overview

Protocol

<img title="figure-results-58" alt="figure-results-58" src="/files/ftp_upload/20169/20169tbl1.jpg" /> 
Table 1. Comparison of the standard and improved protocols for extrachromosomal arrays integration into the&#160;C. elegans&#160;genome.&#160;Two nonintegrated transgenes transmitting at about the same frequency were integrated using the standard or the improved protocol. The main difference lies in the fact that in the standard protocol a screen of the proge...

Results

<table><tbody><tr><td>U.V. cross-linker</td><td>Fischer Scientific</td><td></td><td>Wavelength 254 nm</td></tr><tr><td>Microscope SteREO Discovery V8</td><td>Carl Zeiss, Inc.</td><td></td><td></td></tr><tr><td>Petri dishes, 60 mm</td><td>CML</td><td>BPS55E6</td><td></td></tr><tr><td>Platinium wire 0.25 mm</td><td>Alfa Aesar</td><td>6/4/7440</td><td>To make a pick for worms&nbsp;</td></tr></tbody></table>

UV Irradiation: A Method to Integrate Extrachromosomal Arrays

本文内容

Overview

研究方案

结果

材料

参考文献

Name	Company	Catalog Number	Comments
U.V. cross-linker	Fischer Scientific		Wavelength 254 nm
Microscope SteREO Discovery V8	Carl Zeiss, Inc.
Petri dishes, 60 mm	CML	BPS55E6
Platinium wire 0.25 mm	Alfa Aesar	6/4/7440	To make a pick for worms