L4440

L4440

L4440载体基本信息：

L4440载体图谱：

L4440载体简介：

Reverse Genetics and RNAi：
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 been  particularly well studied in  C. 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. coli  strain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence. HT115 is an RNase III-deficient  E. coli  strain 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. Carbenicillin  is preferred over ampicillin because it tends to be more stable.
Protocol
A. Preparing feeding plates 
1) Inocluate 3mL of LB containing 50 µg/mL  ampicillin with individual desired bacterial strain. Grow overnight at 37oC.
2) Seed NGM agar feeding plates  (containing 25 µg/mL  carbenicillin  and 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. Transfer N2 L4 worms 
1) Transfer two L4 hermaphrodites  from the N2  stock plate to each of the RNAi  feeding 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. Scoring RNAi phenotypes
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 phenotypes  for the experimental RNAi construct and record all your observations in your notebook. Examine  how the observed phenotypes  compare with the corresponding null mutant phenotypes  in the same gene (the WormBase database has information on previously characterized RNAi and null mutant phenotypes).

L4440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 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc

      781 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta

      841 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc

      901 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg

      961 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc

     1021 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt

     1081 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct

     1141 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac

     1201 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact

     1261 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa

     1321 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga

     1381 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt

     1441 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga

     1501 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg

     1561 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat

     1621 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat

     1681 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc

     1741 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga

     1801 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc

     1861 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag

     1921 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc

     1981 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt

     2041 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt

     2101 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat

     2161 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc

     2221 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa

     2281 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg

     2341 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag

     2401 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag

     2461 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa

     2521 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt

     2581 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg

     2641 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc

     2701 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac

     2761 cgagcgcagc gagtcagtga gcgaggaagc

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