1. A Transgenic Approach to QTL analysis in a Trypanotolerant Mouse Model Anderson SI 1 Noyes HA 2 Agaba M 3 Ogugo M 3 Kemp SJ 2,3 Archibald AL 1 1 Roslin Institute (Edinburgh) Roslin Midlothian EH25 9PS UK 2 School of Biological Sciences University of Liverpool Crown Street Liverpool L69 7ZB UK 3 International Livestock Research Institute, P.O. Box 30709 Nairobi 00100 Kenya Acknowledgements: We thank staff at the Roslin Small Animal Unit for microinjections and care of animals. This work was supported by the Wellcome Trust. Introduction We are using a mouse model to investigate the genetic basis of trypanotolerance in African cattle. We have used a transgenic approach in order to evaluate candidate genes underlying a Quantitative Trait Locus (QTL) for trypanotolerance (Tir1) in mice identified in an earlier study. BAC clones from the trypanotolerant C57BL/6 underlying the Tir1 QTL for response to infection with Trypanosoma congolense were microinjected into F1 A/J x Balb/c (susceptible) donor oocytes fertilised with an A/J male. Transgenic animals were repeatedly backcrossed to A/J with genotyping for the transgene at every generation, and finally intercrossed prior to trypanosome challenge. TRA mice TRA (Trypanosome Resistance A) mice were made with NotI linear fragments from BAC clone RP23-27d5. Transgenic mice were identified by the presence of the C57 allele of the D17Mit62 microsatellite. The fragment integrated in the TRA2 founder was shown by SNP analysis to carry and express the C57 allele of Rab11b, but not March2. Ensembl view showing gene content of RP23-27d5 BAC TRB mice TRB mice were made with NotI linear fragments from BAC clone RP24-63k8. Transgenic mice were identified by the presence of the C57 allele of the D17Mit16 microsatellite. The fragment integrated in the TRB founder was shown by SNP analysis to carry and express the C57 allele of Myo1f, but not Adamts10, Zfp414, Pram1 or March2. Ensembl view showing gene content of RP24-27d5 BAC G4 Transgenic mice and non-transgenic sibling controls were infected with Trypanosoma congolense and % survival plotted against survival time (days post-infection). TRA transgenics showed a slight increase in survival at 95 days post-infection. TRA_AA (non-transgenic) TRA_AC (one copy of transgene) TRA_CC (two copies of transgene) TRB_AA (non-transgenic) TRB_AC and _CC (transgenic) Results and conclusions This method enables the dissection of large QTL areas in order to assess the effect of candidate genes, however transgene content of these mouse lines suggests that introducing linear BAC DNA by microinjection results in truncation of the BAC DNA by either shearing and/or recombination. We have successfully created BAC transgenic mice in a strain not commonly used for microinjection by manipulating factors influencing oocyte quality. A slight increase in % survival at day 95 is seen in the TRA line. This is stronger in the mice homozygous for the transgene. SNP genotyping is underway to investigate a possible heterozygote effect on survival time. The TRB line showed no variation in survival time compared to the non-transgenic control, which may rule out the genes carried by this BAC as candidates. 0-40 41-50 51-60 61-70 71-80 81-90 91-100 Days post-infection 0-40 41-50 51-60 61-70 71-80 81-90 91-100 Days post-infection % survival