Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

This protocol fills a technical gap for the biochemical study of C.elegans and expands their usefulness as a model organism. This technique is both simple and robust, allowing for consistent isolation of functionally active C.elegans nuclear extract. Begin by placing the synchronized L1 animals on 10 150-millimeter nematode growth media-containing plates seeded with Escherichia coli OP50, and allow the animals to grow for 48 hours at 20 degrees Celsius until they reach the L4 stage.

Once the hypotonic and hypertonic buffers are prepared, clean the Balch homogenizer by flooding the grinding chamber with 70%ethanol. Then rinse the chamber with deionized water to remove the excess ethanol. Insert the tungsten carbide ball into the grinding chamber.

Cap the ends of the Balch homogenizer's barrel, and secure the caps with the provided thumbscrews. Then prepare five milliliters of the complete hypotonic and hypertonic buffers per sample as described in the text manuscript. Next fill a sterile, two-milliliter syringe with one milliliter of complete hypotonic buffer, and gently flush the grinding chamber of the Balch homogenizer, leaving approximately 500 microliters of complete hypotonic buffer in the chamber.

Store the flushed homogenizer on ice, and let it cool for 30 minutes. Collect the well-fed L4 animals with M9 buffer in a 15-milliliter conical tube, and centrifuge the animals at 1, 000 times g for three minutes. Remove the supernatant, and continue washing the animal pellet until the supernatant is clear.

Next wash the animals with three milliliters of cold hypotonic buffer, and centrifuge again as demonstrated. After removing the supernatant hypotonic buffer, add one milliliter of complete hypotonic buffer to the animal pellet, and transfer the animal suspension to a new sterile, two-milliliter syringe. For homogenizing the animals kept on ice, gently push the animals through the grinding chamber of the Balch homogenizer loaded with the tungsten ball.

Then collect the animals back in the syringe, and repeat this procedure 30 times. After 30 cycles of homogenization, collect maximum animal suspension from the Balch homogenizer, and store the syringe with the tip positioned downward inside a 1.7-milliliter microtube. Remove the tungsten ball, and clean the grinding chamber with deionized water.

Dry and return the ball to the respective labeled tube. Then insert the tungsten ball with 7.9880-millimeters diameter and 12-micrometer gap clearance into the grinding chamber, and reseal the homogenizer. Flush the grinding chamber again with one milliliter of ice-cold complete hypotonic buffer.

Grind the suspension 25 times as demonstrated. Transfer the animal suspension into a clean, 1.7-milliliter microtube, and store the suspension on ice. Pellet down the animal bodies and debris by centrifugation.

Pipette 40 microliters of the supernatant to a tube labeled as input fraction, and store the fraction on ice. Transfer the remaining supernatant to a new 1.7-milliliter tube without disturbing the pellet, and centrifuge to pellet the nuclei. Then transfer the supernatant without disturbing the pelleted nuclei to a new 1.7-milliliter tube labeled as cytosolic fraction.

To wash the nuclei pellet, add 500 microliters of complete hypotonic buffer to the pellet, suspend the pellet, and centrifuge at 4, 000 times g at four degrees Celsius for five minutes. At the end of the centrifugation, resuspend the nuclear pellet in 500 microliters of fresh complete hypotonic buffer, and again centrifuge the sample as demonstrated. Then dissolve the pellet in 40 microliters of complete hypertonic buffer.

Transfer the nuclear suspension to a new 1.7-milliliter tube labeled nuclear fraction, and store it on ice. Determine the protein concentration of three fractions using a fluorescent quantification kit. Aliquot the nuclear fractions into single-use tubes containing six micrograms of the nuclear protein, and snap freeze in dry ice and ethanol bath.

Store the samples at minus 80 degrees Celsius until further use. The representative gel image shows the transcription products of Caenorhabditis elegans L4 larvae nuclear extract using the CMV promoter DNA template. The successful isolation of active nuclear proteins resulted in a 132 base pair band after an in vitro transcription, and unsuccessful isolation resulted in a weak band or the absence of a band.

Remember to clearly label the complete buffers. Inappropriate buffers can harm the functionality of the nuclear proteins. Also, keep the homogenizer ice cold to prevent denaturing of the proteins.

Developing this technique allowed researchers to measure the rate of RNA transcription of C.elegans during stressful conditions, showing that the animal's transcription rate can change depending on environmental conditions.