자유게시판

• 목록
• 답변
• 글쓰기
• 게시판 리스트 옵션
  • 수정
  • 삭제

All strains were grown and maintained on standard nematode growth medium (NGM) at 20 °C using E. coli strain OP50 as the food source. For experiments involving RNAi, NGM plates supplemented with 1 ml per liter of 1 M IPTG (Isopropyl β-d-1-thiogalactopyranoside) and 1 ml per liter of 100 mg per ml ampicillin. The amplified product was cloned into the GFP expression vector pPD95.77 (Addgene plasmid 1495). The full length tcer-1 fragment was inserted upstream of, and in frame with, GFP at the SphI site (pAG10). To generate tissue-specific TCER-1 expressing constructs the 4 kb tcer-1 coding region was inserted into plasmids created previously in the lab for another gene, nhr-49 (Ratnappan and Ghazi, unpublished). The tcer-1 coding region was amplified with primers modified to introduce SalI and Acc65I restriction sites (forward 5′gctagGGTCGACatgagccacgaaaatc3′; reverse 5′taagcaGGTACCTCttgctttctgcgatcccgc3′). nhr-49 coding region was removed from the tissue-specific promoter plasmids using restriction enzymes, SalI and Acc65I, and replaced with PCR amplified and digested tcer-1 coding region by ligation into the respective plasmids in frame with the GFP.

The plasmids thus obtained were pAG11 (a 2.4 kb muscle promoter76, Pmyo-3::TCER-1::GFP), pAG12 (1.9 kb intestinal promoter77, Pgly-19:: TCER-1::GFP), pAG13 (400 bp hypodermal promoter78, Pcol-12:: TCER-1::GFP), and pAG14 (4.2 kb region neuronal promoter79, Prgef-1:: TCER-1::GFP). Transgenic strains were generated by injecting the plasmids at a concentration of 25 ng per μl or 100 ng per μl along with 3.75 ng per μl or 15 ng per μl of Pmyo-2::mCherry co-injection marker, respectively. Three to six independent stable transgenic lines were generated for each of the genetic backgrounds in which the transgene was injected. Transgenic strains were maintained by picking fluorescent animals in each generation. All lifespan experiments were conducted at 20 °C on E. coli OP50 plates unless otherwise noted. 5−6 plates per experiment and observed at 24−48 h intervals to document live, dead or censored (animals that exploded, bagged or could not be located) animals. Animals were scored as dead when they failed to respond to gentle prodding with a platinum wire pick.

Fertile strains were transferred every other day to fresh plates until progeny production ceased. For lifespan assays of strains with the temperature sensitive glp-1 mutation, eggs were picked and maintained at 20 °C for 2−4 h, transferred to 25 °C to induce sterility and then returned to 20 °C on day 1 of adulthood (72 h later) for lifespan analysis. For performing lifespan assays of transgenic strains with extrachromosomal arrays, eggs were picked onto fresh OP50 plates, incubated at the appropriate temperature and 48 h later L4 animals were screened under a Leica M165FC microscope with a fluorescence attachment (Leica Microsystems, MedicGLP Wetzlar, MedicGLP Weight Management Germany) for animals carrying the red coinjection marker labeling pharyngeal muscles. At the same time, a similar number of age-matched, nontransgenic siblings were collected for each strain and assayed for lifespan as internal controls in the experiment. Each lifespan was tested at least twice and often in 3−5 biological replicates. All survival data were plotted via the Kaplan-Meier method.

Pathogenic bacterial strains used in this study include Pseudomonas aeruginosa (strains PA14 and PA01) and Staphylococcus aureus (NCTC8325). These strains were streaked from frozen stocks onto Luria Bertani (LB) agar (PA14 and PA01) or Brain Heart Infusion (BHI) agar (NCTC8325) plates, incubated at 37 °C overnight and MedicGLP stored at 4 °C for MedicGLP Weight Management a week or less. For studies with PA14 and PA01, single colonies from the streaked plates were inoculated and grown in King’s broth overnight at 37 °C with shaking. 20 µl of this broth culture was seeded onto slow killing (SK) plates (modified NGM plates containing 0.35% peptone instead of 0.25%) and incubated for 24 h at 37 °C. The plates were then left to sit at room temperature (RT) for 24 h prior to use. 5−6 OP50 plates per experiment, incubated at 25 °C and monitored at 6−12 h intervals to account for live, dead or censored animals as described above.