l4440使用说明资料.pdf

L4440L4440 编号编号载体名称载体名称 北京华越洋北京华越洋 VECT75473VECT75473L4440L4440 L4440L4440 载体载体基本信息：基本信息： 启动子启动子: :T7 复制子复制子: :pUC ori，F1 ori 质粒分类质粒分类: :线虫 RNAI 干扰载体；Worm Expression RNAi 质粒大小质粒大小: :2790bp 质粒标签质粒标签: :lacZN, OriF1 原核抗性原核抗性: :氨苄青霉素 Ampicillin 克隆菌株克隆菌株: :DH5α 培养条件培养条件: :37℃，有氧，LB 表达宿主表达宿主: :线虫，worm 诱导方式诱导方式: :IPTG 5'5'测序引物测序引物: :根据全序列设计 L4440L4440 载体图谱：载体图谱： L4440L4440 载体简介：载体简介： ReverseReverse GeneticsGenetics andand RNAiRNAi：： RNAi is a natural cellular process in many eukaryotes which scientists have taken advantage of in the lab as a valuable reverse genetics mechanism for regulating gene expression. RNAi involves double-stranded RNA (dsRNA) interfering with the expression of genes with sequences complementary to the dsRNA. By administering a specific dsRNA to a cell culture or multicellular organism, RNAi can be induced to selectively reduce expression of that specific gene in the cells or organism. RNAi has beenparticularly well studied inC. elegans, in which the RNAi gene silencing phenotype is heritable and the dsRNA is easily administered.E. coli bacteria carrying RNAi plasmids that produce the desired dsRNA can be fed to worms, and the dsRNA is transferred to the worm via the intestinal tract. RNAi plasmids typically consist of DNA coding sequence from the intended target gene cloned between two T7lac promoters. The plasmid also has a selectable marker that confers resistance to an antibiotic, in this case ampicillin. In 7.15, you will use the E. colistrain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence. HT115 is an RNase III-deficientE. colistrain with IPTG-inducible T7 polymerase activity.To induce dsRNA production from these plasmids, the HT115 bacteria is grown on special RNAi NGM feeding plates that contain IPTG and the ampicillin analog carbenicillin. Carbenicillinis preferred over ampicillin because it tends to be more stable. ProtocolProtocol A.A. PreparingPreparing feedingfeeding platesplates 1) Inocluate 3mL of LB containing 50 µg/mLampicillin with individual desired bacterial strain. Grow overnight at 37oC. 2) Seed NGM agar feeding plates(containing 25 µg/mLcarbenicillinand 1mM IPTG) by pipetting 150 µL of LB bacterial culture into the center of the plate.You should have three RNAi feeding plates: a. One plate will be seeded with HT115 bacteria carrying the “empty” L4440 vector (no C. elegans gene is cloned in the vector). b. Another plate will be seeded with HT115 bacteria carrying a portion of the unc-22 gene cloned into the L4440 vector. The phenotype resulting from RNAi of unc-22 is well-established and is reliably reproducible under the experimental conditions you are using today. c. A third plate will be seeded with HT115 bacteria carrying a portion of your gene of interest cloned into the L4440 vector.We will use dpy10 as our gene of interest. 3) Let the plates dry overnight at room temperature. B.B. TransferTransfer N2N2 L4L4 wormsworms 1) Transfer two L4 hermaphroditesfrom the N2stock plate to each of the RNAifeeding plates, minimizing the amount of OP50 bacteria transferred. Try to avoid bringing any younger contaminating worms along with the L4 worms you are transferring. 2) Incubate the plates until the progeny reach the desired age (Note: it takes ~4 days for a C. elegans egg to grow into a gravid adult when grown at 16oC. C.C. ScoringScoring RNAiRNAi phenotypesphenotypes 1) Observe RNAi controls. Start by looking at the L4440 RNAi plates- what phenotype(s) would you expect to see on these plates? Next, look at the unc-22 RNAi plates – what phenotype(s) would you expect to see on these plates? Record all your observations in your notebook. If you do not see the expected phenotypes on your control plates, this may indicate your RNAi experiment was set-up incorrectly. 2) Observe the RNAi phenotypesfor the experimental RNAi construct and record all your observations in your notebook. Examinehow the observed phenotypescompare with the corresponding null mutant phenotypesin the same gene (the WormBase database has information on previously characterized RNAi and null mutant phenotypes). L4440L4440 载体序列：载体序列： ORIGIN 1 aacctggctt atcgaaatta atacgactca ctatagggag accggcagat ctgatatcat 61 cgatgaattc gagctccacc gcggtggcgg ccgctctaga actagtggat ccaccggttc 121 catggctagc cacgtgacgc gtggatcccc cgggctgcag gaattcgata tcaagcttat 181 cgataccgtc gacctcgagg gggggcccgg tacccaattc gccctatagt gagtcgtatt 241 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 301 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 361 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta 421 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 481 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 541 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 601 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 661 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 721 a。