이 프로토콜은 Kolundzic, 외,<a href="https://www.jove.com/video/56889/application-rnai-heat-shock-induced-transcription-factor-expression">RNAi및 열 충격 유도 전사 인자 발현의 응용 프로그램에서 발췌하여 생식세포를 C. 엘레건의뉴런으로 재프로그래밍하기 위해,</a> J. Vis. Exp. (2018). 
<ol><li>솔루션 준비
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NGM-아가 플레이트 (1 L)
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<li>NaCl 3g, 펩톤 미디어 2.5g(예: 바토-?...

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

출처: 콜룬드치치, E., 외. <a href="https://www.jove.com/video/56889/application-rnai-heat-shock-induced-transcription-factor-expression">RNAi 및 열 충격 유발 전사 인자 발현의 적용은 C. elegans에서뉴런에 세균 세포를 다시 프로그래밍한다.</a> J. 비스 익스펙. (2018).
이 비디오는 C. elegans에서 RNAi 처리의 뒤에 원리를 설명하고 형질 전환벌레 긴장에 있는 lin-35를 노크하는 프로토콜을 보여줍니다.

<img alt="Figure 1" src="/files/ftp_upload/20138/20138fig1.jpg"> 그림 1: 린-53에 대한 RNAi에 의해 발생 Pvul 표현형. (상단) L4/젊은 성인 단계 F1 자손 웜의 DIC 사진 제어 또는 lin-53 RNAi 치료 어머니에서 파생 된. 스케일 바 = 20 μm. (아래) lin-53 RNAi로 처리된 동물은 돌출된외음부(pvul)표현형(블랙 애로우 헤드)을 표시한다. pvul 표현형은 린-53에 대?...

C. 예르간 먹이RNAi 도금: 대장균에서 표적 dsRNA 발현을 유도하는 기술

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.