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Congenic mice for the identification of genes responding to infection with Trypanosoma congolense. Noyes HA 1 Agaba M 2 Gibson J 3 Ogugo M 2 Iraqi F 2 Brass A 4 Anderson S 5 Zeef L 4 Hulme H 4 Kemp SJ 1 1 School of Biological Sciences, University of Liverpool, Crown Street,

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Congenic mice for the identification of genes responding to infection with Trypanosoma congolense

Noyes HA1

Agaba M2

Gibson J3

Ogugo M2

Iraqi F2

Brass A4

Anderson S5

Zeef L4

Hulme H4

Kemp SJ1

1School of Biological Sciences,

University of Liverpool,

Crown Street,

Liverpool L69 7ZB

2International Livestock Research

Institute (ILRI), P O Box 30709,

Nairobi, 00100 Kenya

3The Institute for Genetics &

Bioinformatics, Hawkins Homestead,

University of New England,

Armidale, NSW 2351,

Australia

4Department of Computer Science,

University of Manchester,

Oxford Road,

Manchester M13 9PL

5Roslin Institute, Roslin BioCentre,

Midlothian EH25 9PS

Introduction

Trypanosoma congolense a protozoan parasite transmitted by tsetse flies causes nagana, a sleeping sickness like disease, in cattle throughout much of Sub-Saharan Africa. A mouse model of genetic control of trypanotolerance exists based on A/J as a susceptible strain and C57BL/6 as a tolerant strain. Five major trypanotolerance QTL have been identified on mouse chromosomes 1, 5, and 17, associated with survival time (Iraqi and Clapcott et al 2000). We have developed three congenic mouse lines that carry regions derived from C57BL/6 that cover the trypanotolerance QTL on an A/J background. The carriers of the C57BL/6 DNA for the QTL survive longer after infection than non-carrier litter-mate controls. The difference in survival time is similar to that predicted from the original mapping experiments.

Thirteen congenic and control mice were genotyped using the Illumina 1500SNP mapping panel in order to identify the positions of the C57BL/6 regions in the genomes. This data identified a number of regions carrying C57BL/6 origin DNA that were outside the trypanotolerance QTL. QTL for other phenotypes that overlap these regions were identified. These mice could be developed into congenic lines for these phenotypes within 2-3 generations and we will be happy to supply them to interested academic investigators.

Iraqi F, Clapcott SJ, Kumari P, Haley CS, Kemp SJ, Teale AJ. Fine mapping of trypanosomiasis resistance loci in murine advanced intercross lines. Mamm Genome. 2000 11:645-8.

Congenic Mice carry C57BL/6 regions on an A/J bacground that overlap multiple QTL

Location of Trypanotolerance QTL and regions containing C57BL/6 DNA on an A/J background.

Five QTL for survival time after infection with Trypanosoma congolense have been mapped to high resolution in F6 advanced intercross lines and named Trypanosoma infection response (Tir). C57BL/6 carried alleles for longer survival time at all these loci, are indicated by red arrows in the figure.

Three congenic mice lines were generated by crossing C57BL/6 x A/J and backcrossing to A/J after selection for carriers of the C57BL/6 allele at each of three QTL. Mice were backcrossed for six generations. The backcrossed mice were then intercrossed and homozygote carriers and non carriers for each of the QTL were selected for genotyping and phenotyping. 13 mice were genotyped using the Illumina 1500 SNP mouse genotyping panel of which approximately 800 SNP are informative between A/J and C57BL/6. The mice were found to carry regions of 12-40Mb that overlapped the QTL and additional regions external to the QTL. These are shown in the figure by bars of solid colour.

These off target regions provide a rich resource for developing further congenic lines to fine map phenotypes for other traits that have already been mapped to those regions. A list of QTL that have been mapped to regions overlapping the C57BL/6 regions of the congenic lines is shown in the right hand panel. Intriguingly this list includes three QTL for response to infection with the related protozoan Leishmania major, as well as Babesia and Plasmodium chaboudi.

Congenic mice carrying C57BL/6 alleles at QTL survive longer than littermate controls

The three congenic mice lines that carried the C57BL/6 (resistant) alleles at each QTL survived longer than their littermate controls that carried the A/J alleles at these loci. In each case the difference in survival time was similar to the difference predicted by the original mapping studies confirming that there is no evidence for interactions between QTL. There was evidence for substantial heterosis since littermate controls that carried A/J alleles at the QTL still survived about twice as long as the parental inbred A/J mice.

Only a single QTL was identified on chromosome 1 in the original F2 mapping population which was designated Tir3. In the subsequent F6 mapping population three QTL were mapped to the same region and designated Tir3a, Tir3b and Tir3c. The congenic interval only spanned Tir3a and Tir3b, however the difference in survival time was close to that attributed to all three Tir3 loci. Consequently it is possible that the Tir3c QTL is an artefact.

Evidence for trans regulation of genes outside congenic regions

Affymetrix 430_2 arrays were used to determine expression of all genes in the liver. The log ratio of expression genes of carriers of C57BL/6 alleles to expression from littermate non-carriers was determined and plotted against chromosomal position. The upper figure shows the ratio of expression of genes on chromosome 17 of the Tir1 congenics which carry C57BL6 alleles in the 26-44Mb region. A cluster of extreme ratios can be seen in this region. This is the region with largest expression ratios in the genome, however some genes on other chromosomes also have higher than expected expression ratios.

The lower figure shows expression ratios on chromosome 5. CXCL1 at 92Mb is more than twofold over expressed in the carriers of C57BL/6 alleles at the Tir1 locus on chromosome 17. Resequencing confirmed that the congenic mice carried A/J alleles at this locus. This data suggests that CXCL1 is trans regulated by a gene in the chromosome 17 QTL region, although it may be regulated by the other congenic regions on other chromosomes. 14 other genes across the genome and not in congenic regions were over two fold under or over expressed and also may be trans regulated.

CXCL1