Dieses Protokoll ist aus Kolundzic, et al, <a href="https://www.jove.com/video/56889/application-rnai-heat-shock-induced-transcription-factor-expression">Application of RNAi and Heat-shock-induced Transcription Factor Expression zur Neuprogrammierung von Keimzellen auf Neuronen in C. elegans</a>, J. Vis. Exp. (2018).
<ol><li>Lösungsvorbereitung
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NGM-Agar Platten (1 L)
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<li>Fügen Sie 3 g NaCl, 2,5 g Peptonmedien (z. B. Bacto-Peptone) und ...

rnai plating for c. elegans feeding: a technique to induce target dsrna expression in e. coli

Quelle: Kolundzic, E., et al. <a href="https://www.jove.com/video/56889/application-rnai-heat-shock-induced-transcription-factor-expression">Anwendung von RNAi und Hitzeschock-induzierten Transkriptionsfaktor-Expression, um Keimzellen auf Neuronen in C. elegans umprogrammieren.</a> J. Vis. (2018).
Dieses Video beschreibt die Prinzipien hinter der RNAi-Behandlung in C. elegans und zeigt ein Protokoll zum Knockdown von lin-35 in einem transgenen Wurmstamm.

<img alt="Figure 1" src="/files/ftp_upload/20138/20138fig1.jpg"> Abbildung 1: Pvul-Phänotyp, der durch RNAi gegen Lin-53 verursacht wird. (Oben) DIC-Bild von L4/jungen Erwachsenen Stadium F1 Nachkommenwürmer von Kontrolle abgeleitet oder lin-53 RNAi behandelt Mütter. Skalenstäbe = 20 m. (Unten) Tiere, die mit lin-53 RNAi behandelt werden, zeigen den hervorstehenden Vulva (Pvul) Phänotyp (schwarze Pfeilköpfe). Der Pvul-Phänoty...

RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli

Source: Kolundzic, E., et al. Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in&#160;C. ...

- In C. elegans, RNAi can be induced by feeding worms bacteria that express double-stranded RNA or dsRNA from a recombinant DNA plasmid. To begin, grow tetracycline-resistant HT115 E. coli transfected with L4440 plasmid overnight on selective LB agar plates containing tetracycline and ampicillin, or its functional analog carbenicillin. In addition to the target DNA, the plasmid contains a gene for ampicillin resistance. Only bacteria colonies that inherit the plasmid can grow in the presence of these antibiotics. Next, harvest bacteria colonies and expand them in liquid LB medium to the desired concentration. To induce dsRNA expression, transfer the bacteria solution onto prepared nematode growth medium, or NGM, agar plates containing IPTG. In this expression system, IPTG mimics lactose to inactivate the lac repressor enabling the expression of T7 RNA polymerase. On the plasmid, target DNA expression is regulated by two convergent T7 promoters. Consequently, sense and anti-sense sequences are transcribed leading to the expression of dsRNA. Finally, the worm uses the sequence information from the ingested dsRNA to downregulate endogenous mRNAs with complementary sequences. In this experiment, we will prepare RNAi plates with transgenic E. coli for C. elegans feeding. - For the RNAi plates, load 6-centimeter plates with NGM agar containing IPTG and carbenicillin. Let them dry for 24 to 48 hours at room temperature in the dark. Then, transfer them to 4 degrees Celsius for up to 14 days. Next, streak selective plates containing carbenicillin and tetracycline with RNAi bacteria clones with L4440 plasmid carrying the lin-53 gene. Use a three-phase streaking pattern and be sure to plate an empty vector control. Then, grow the plates overnight at 37 degrees Celsius. The next day, pick at least three single colonies and inoculate each of them into a separate culture tube with 2 milliliters of LB supplemented with carbenicillin, but not tetracycline. Plan to have three healthy cultures per condition. Next, grow the cultures overnight at 37 degrees Celsius until they reach an optical density at 600 nanometers of 0.6 to 0.8. Then, add 500 microliters of each bacterial culture to a 6-centimeter NGM agar RNAi plate. Incubate the plates overnight at room temperature in the dark.