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Defining a Role for SARM in Pathogen Resistance and PA14 Unigene Library Construction

Defining a Role for SARM in Pathogen Resistance and PA14 Unigene Library Construction Nicole T. Liberati Group Meeting 6/10/02. Requirement for a p38 MAP kinase signaling pathway in C. elegans immunity. PATHOGEN. ???. ESP-8 (MAPKKK). ESP-2 (MAPKK). PMK-1 (p38 MAPK). ???.

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Defining a Role for SARM in Pathogen Resistance and PA14 Unigene Library Construction

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  1. Defining a Role for SARM in Pathogen Resistance and PA14 Unigene Library Construction Nicole T. Liberati Group Meeting 6/10/02

  2. Requirement for a p38 MAP kinase signaling pathway in C. elegans immunity PATHOGEN ??? ESP-8 (MAPKKK) ESP-2 (MAPKK) PMK-1 (p38 MAPK) ??? ??? IMMUNE RESPONSE

  3. PA14 Slow Killing Assay

  4. Longevity Assay

  5. Slow Killing Assay at 20°C

  6. Genomic Structure of F13B10.1

  7. SARM Long SARM Short

  8. ceSARM dSARM hSARM mSARM

  9. No C. elegans or D. melanogaster mutants available SARM Expression patterns C. elegans: expressed at all ages - highest in oocytes and embryos D. Melanogaster: (Ect4) a) repressed by Dorsal b) expressed in the dorsal ectoderm H. sapiens: Multiple Tissue Northern

  10. Mink et al Genomics(2001) 74:234-44

  11. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  12. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  13. Mouse adapter protein northern blots BMDM DC RAW264.7 BMDM DC RAW264.7 ___ ___ ___ - + - + - + LPS ___ ___ ___ - + - + - + LPS TRIF SARM b-actin MyD88 TRAM b-actin MAL gives very faint hardly detectable bands H. Byorkbaka and M. Freeman

  14. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  15. TLR2/1 or 2/6 TLR3 TLR4 TLR5/7/8/9 MD2 ? MyD88 MyD88? TRIF/ Mal MyD88 ? MyD88 TICAM1 Mal ? NFkB IRF3 NFkB NFkB NFkB IRF3 TNFa IFNb IL1 RANTES IL6 IL8 TNFa IL1 IL6 IL8 TNFa IFNb IL1 RANTES IL6 IL8 TNFa IL1 IL6 IL8 O’Neill and Fitzgerald

  16. Cloning the human SARM cDNA IMAGE clone EagI EagI Corrected 240 bp PCR product Cloned into pBSKII+ to generate hSARM 240/pBSKII+ EagI A partial EagI/NheI IMAGE clone Fragment cloned into hSARM240/pBSKII+ to generate hSARM/pBSKII+ EagI NheI PCR products of full length cDNA subcloned into pUNI system and FLAG fusion vector.

  17. TIR domain Crystal Structure TLR2 TIR consensus TLR5 hSARM

  18. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  19. Targeted Deletion Strategy 5’ arm 3’ arm neo TK Transfect C129 ES cells with targeting construct Positive selection (neo resistance) and Negative selection (thymidine kinase) Southern screen for integrated selection marker Inject mutant ES cells into Black6 blastocyst generating chimeric embryos that are introduced into foster mothers Search for germ line transmission among offspring of chimeric founders Breed heterzygous offspring to generate homozygous null progeny-PCR screening

  20. SARM Mouse Targeted Deletion Mutant Strategy 15kb WT E E 1 2 3 4 5 6 7 8 9 MT E E 1 neo 8 kb

  21. 200 pg control 200 pg control 20 pg control 20 pg control Ladder HindIII Ladder BamHI HindIII BamHI EcoRI EcoRI

  22. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  23. RETROVIRAL INSERTIONAL MUTAGENESIS in diploid loci paternal maternal + mutagenic virus heterozygous at viral insertion site no phenotype induction of MR/ non-disjunction selection for homozygosity at integration site spontaneous LOH (loss of heterozygosity) homozygous at viral insertion site phenotype .. selection for phenotype locate retroviral insertion site to identify disrupted gene responsible for phenotype

  24. Independent Recombination Between LoxP Variants loxP* loxP loxP loxP* R1 IRES TK R2 LOH loxP loxP* loxP* loxP R1 R2 IRES TK R1 R2 IRES TK Tat - cre R1 R1 R2 R2 R1 R2 R1 R2 R1 +, R2 + , TK - R1 R2 R2 R1 selection for phenotype

  25. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  26. Phospho-p38 Western of RNAi-treated worm lysates RNAi Vector Control RNAi Vector Control F13B10.1 RNAi + Control lysate pmk RNAi pmk RNAi

  27. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  28. C. Elegans Deletion Screen Overview A/B PCR Screen PspGI C/D PCR Screen PspGI PspGI

  29. Deletion Screen Overview + + - - PspG1 C/D C/D A/B A/B

  30. + - + - + - PspGI

  31. Sequencing: a) correct part of genome b) quality poor

  32. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  33. F13B10.1 GFP Expression Constructs

  34. hSARM Experimental Overview Mammalian Studies 1) siRNA in immune cells 2) hSARM overexpression in TLR/NFKB/IRF3 reporter assays 3) hSARM targeted deletion in mice 4) hSARM homozygous null Embryonic Stem cells C. elegans Studies 1) phospho-p38 Western 2) F13B10.1 deletion screen 3) GFP expression study 4) F13B10.1 overexpression analysis WT or TIR mutant rescue esp phenotype in RNAi-treated worms DK’s sek-1 reporter strains/p38 western 5) Interactome

  35. Prosite: nuclear localization; Similarity to P. furiosus glyceraldehyde 3-phosphate dehydrogenase SARM Interactors Mkk4 interactor Personal communication, Pierrot-Vidalain and M. Vidal

  36. Acknowledgements Ausubel Lab Dennis Kim Rhonda Feinbaum Dan Lee Jas Villanueva Sachiko Miyata Jonathan Urbach Tao Wei Rubin Lab Chris Sassetti Mekalanos Lab Gene Targeting Facility Freeman Lab Scott Kim Harry Byörkbacka Francois Golenbock Lab Kate Fitzgerald Vidal Lab Pierrot Olivier Ruvkun Lab Sylvia Lee Ho Yi Wei Ching Seed Lab Naifang Lu Yi Yang

  37. TraSH Methodology: Probe Production 5) Reverse Transcription with fluorophore to produce labeled cDNA 6) Hybridize differentially labeled probe pools to microarray Sassetti, C. M., Boyd, D. H., and Rubin, E. J., (2001). PNAS 9812712-12717

  38. TraSH Methodology: Detection of the Presence or Absence of Tn Mutants in a Pool of Mutants Sassetti, C. M., Boyd, D. H., and Rubin, E. J., (2001). PNAS 9812712-12717

  39. pMFLGM.GB- 2xT7 (pMAR 2XT7) lacZ Mariner Transposase pir-dependent ori 29 bp IR T7 Promoter pMAR 2xT7 Gm resistance T7 Promoter Tra genes 29 bp IR Amp resistance Inserted into the PA14 genome:

  40. TraSH PCR products 1 primer 2 primers Mar1xT7 DNA Mar1xT7 DNA Mutant 1 Mutant 1 Mutant 5 Mutant 2 Mutant 3 Mutant 4 Mutant 5 Mutant 2 Mutant 3 Mutant 4

  41. Mar1xT7 DNA Mar1xT7 RNA Mar1xT7 PCR Mutant 1 + control Mutant 5 Mutant 2 Mutant 3 Mutant 4 2 primer Ladder

  42. 1 primer control 1 primer control Mutant 3 + control Mutant 1 Mutant 4 Mutant 5 Mutant 2

  43. Gentimycin Titration Irgasan Titration

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