Sheep

Coordinator: Gary Entrican

Gary Entrican is Immunology Leader at Moredun Research Institute and the overall Immunological Toolbox Co-ordinator. His contribution to the Toolbox focuses on development of reagents to study cytokine biology in sheep and evaluation of those reagents for cross-reactivity with other species, notably cattle.

Key targets

sheep

  • IL-1b (recombinant protein, mAbs, ELISA)
  • IL-8/CXCL8 (recombinant protein, mAbs, ELISA)
  • IFN-g (quantitative real-time RT-PCR)
  • IL-10 (quantitative real-time RT-PCR)

See progress summary

Other targets

  • MHC Class I (haplotype-specific mAbs)
  • IDO (molecular detection probes)
  • Stable mammalian expression systems for recombinant proteins
  • Production and evaluation of species-cross reactive cytokines within the consortium

See progress summary

 

Progress on key targets for sheep
Updated 22 July 2009

IL-1β
Recombinant ovine IL-1β has been expressed in CHO cells using the pEE14 (Lonza)™ vector. One new mAb has been produced from a mouse immunised with a combination of the CHO-expressed ovine IL-1β and ovine IL-1β expressed in bacteria using the pIVEX™ plasmid. More mice have been immunised with CHO-expressed ovine IL-1β and fusions are planned for autumn 2009 with a view to producing a mAb pair for ELISA development.

CXCL8
Recombinant ovine CXCL8 has been expressed in CHO cells using the pEE14 (Lonza)™ vector. Two new mAbs have been produced from a mouse immunised with a combination of the CHO-expressed ovine CXCL8 and ovine CXCL8 expressed in bacteria using the pIVEX™ plasmid. One of these mAbs reacts with recombinant bovine CXCL8 but not with recombinant equine CXCL8, both of which were provided by our collaborators in US VIRN. This mAb reacts with ovine CXCL8 in fixed tissue (see Wheelhouse et al, 2009 in the publications list). ELISA development is underway.

IFN-γ
Specific molecular probes have been developed for quantitative real-time PCR measurement of mRNA encoding ovine IFN-γ in peripheral blood mononuclear cells stimulated with mitogen and antigen. IFN-γ protein expression has also been measured by ELISA to determine the relative kinetics of mRNA and protein expression. The relative kinetics of IFN-γ and IL-10 expression have also been compared (see below). Manuscript in preparation.    

IL-10
Specific molecular probes have been developed for quantitative real-time PCR measurement of mRNA encoding ovine IL-10 in peripheral blood mononuclear cells stimulated with mitogen and antigen. IL-10 protein expression has also been measured by ELISA to determine the relative kinetics of mRNA and protein expression. The relative kinetics of IL-10 and IFN-γ expression have also been compared (see above). Manuscript in preparation.

   

Progress on other targets for sheep
Updated 22 July 2009

MHC Class I
Mice have been immunised with peripheral blood mononuclear cells from the Moredun flock of MHC class-1 haplotype defined sheep (see Ballingall et al, 2008 in the publications list). The mice were boosted with cells transfected with ovine MHC class 1 genes. Three fusions have been conducted to date, with no haplotype-specific mAbs being developed. Due to the lack of success, this target has been suspended.  

IDO
Ovine IDO has been cloned. PCR primers have been developed to detect IDO expression in IFN-γ activated ovine cells (see Entrican et al, 2009 in the publications list). The cDNA encoding ovine IDO has been cloned into the pEE14 (Lonza)™ vector and expressed in CHO cells.  

Mammalian protein expression
The pEE14 (Lonza)™ vector has been used to stably express a number of biologically-active ovine and bovine cytokines in transfected, cloned CHO cells (http://www.moredun.ac.uk/CytokineList.asp). Other immunoregulatory molecules such as ovine IDO (see above) have also been expressed using this system.  

Evaluation of species-cross reactivity of reagents
To avoid duplication of effort and maximise the application of our tools and reagents, cytokine and mAbs are being checked for species cross-reactivity. The transfected CHO cells have proven invaluable for assessing the cross-reactivity of mAbs. We have identified that only one of the two commercially-available mAbs to bovine TNF-α (CC328) detects intracellular ovine TNF-α, the other does not (CC327). This explains the failure of this mAb pair to detect ovine TNF-α by ELISA, but do successfully detect bovine TNF-α (manuscript submitted).

 

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