cerise “c. elegans rnai space experiment” cerise “c. elegans rnai space experiment” rna...
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CERISECERISE““C. elegans C. elegans RNAi space experiment”RNAi space experiment”
RNA interference and protein phosphorylation in space environment using
the nematode Caenorhabditis elegans.
PI: Atsushi HIGASHITANI
Graduate School of Life Sciences,Tohoku University
E-mail: [email protected] Mar. 26, 2009 @ JAXA
The advantages of working with C. elegans are (1) short life cycle (3.5 days at 20oC), (2) entire genome sequenced in which about 35% of genes have human homologs, and (3) cell lineage identified completely.
Introduction of Introduction of Caenorhabditis elegansCaenorhabditis elegans
The Nobel Prize in Physiology or Medicine 2006. "for their discovery of RNA interference - gene silencing by double-stranded RNA using the nematode C. elegans"
Andrew Z. Fire Craig C. MelloStanford Univ. Univ. of Massachusetts
Medical School
RNA interference: RNAi
nuceli
DNA
Cell
Ribosome: Translation
Protein
“ Targeting” specific gene silencing
mRNA degradation
Protein general
RNAi
Transcription: DNA>mRNA
Science overviewScience overview
RNAi technology performs in lots of life science researches, currently, has been applied several clinical examinations.
However, it hasn’t been verified to work the However, it hasn’t been verified to work the RNAiRNAi in in space. space.
Research objectivesResearch objectives
As objective , we propose to investigate whether Ⅰ RNAi occurs in space as the same manner as on the earth.
EarthEarth
Effects of space radiations
Effects of interaction of space radiations & g
11gg
Effects of g
11ggggSpaceSpace
Effects of g
CBEFCBEF
Credit: JAXA
RNAi for OBJECTIVE ⅠRNAi for OBJECTIVE ⅠTwo target genes to monitor RNAi activity in space • Recombinant GFP (Green fluorescent protein) gene• Ce-RBX-1: an E3 ubiquitin ligase gene
Reduction of green fluorescence by feeding dsRNA of GFP
Ce-RBX-1: an E3 ubiquitin ligase that is essential for meiosis, mitosis, and chromosomal condensation and segregation.
Ce-rbx-1 RNAi
Ce-rbx-1 RNAi
2 cells to 4 cells in WT
OBJECTIVE ⅡOBJECTIVE ⅡEffects of microgravity on entire gene and protein expressions, and protein phosphorylations beyond generations.
• Muscle atrophy and degradation• Signal transduction against stresses under space environments• Epigenetic modification
A: Before activation
B: Activation by removing U-pin in space
2 ml L1 larvae or dauers and 12 ml bacterial feeds
Culture systemCulture system
4 days: 1st generation
activation
8 days: 2nd-3rd generation
12 ml bacterial feeds
separated with U-pin
2 ml C. elegans
4- and 8-days culture at 20oC in space
1st generation: embryogenesis on earth2nd -3rd generation: embryogenesis in space
C: Termination by freezing in MELFI
Culture bag Holder Meas Exp Unit A
16 bags 1 Holder
CBEF
4 Meas Exp Unit A(s)
Bio Dosimeter
Experimental flow for turn overExperimental flow for turn over
Object Method
RNAi Cytological analyses
Real time RT-PCR
Gene and proteinexpression
DNA micro array
Proteome analyses
Western blot analysis
Epigenetic modification
DNA methylation analysis
Space radiations
PADLES(Passive dosimeter for lifescience experiments in space)
Post flight analysisPost flight analysis
TsukubaTsukuba
JAXAJAXAAkira Higashibata Akira Higashibata et al. et al.
PerspectivePerspective
Finally, the experimental results from flight samples might clarify the verification of RNAi activity and stress responses including muscle atrophy and alteration of gene and protein expressions under space environments. We expect that the data from this proposal will be useful for providing physiological and molecular biological effects of microgravity and clinical application of RNAi technology in space.
Credit: JAXA
Credit: JAXA
Project Team MembersProject Team MembersTohoku UniversityJAXATohoku UniversityTohoku UniversityTohoku UniversityJAXAJAXAJAXAAESJSF
Atsushi HigashitaniAkira Higashibata
Mori ChihiroTakako Takanami
Nahoko HigashitaniTakashi YamazakiTomoko Sugimoto
Noriaki IshiokaToko Hashizume
Keiji Fukui
AbbreviationAbbreviation
CBEF: cell biology experiment facilityPADLES: passive dosimeter for lifescience experiments in spaceRNAi: RNA interferenceRT-PCR: reverse transcriptase-polymerase chain reactionTLD: thermoluminescence dosimeter