Title: Functional genomics to identify genes and networks influencing survival following Trypanosome challenge.
1Functional genomics to identify genes and
networks influencing survival following
Trypanosome challenge.
2Shirakawa Institute of Animal Genetics
KARI-TRC
3Origins of NDama and Boran cattle
Boran
NDama
4(No Transcript)
5Mouse model
6- Studying the tolerant/susceptible phenotype has
problems - Separating cause from effect
- Separating relevant from irrelevant.
- Dominance of the what is happening to this
weeks trendy gene/protein/cytokine? approach.
7- Studying tolerance susceptibility any way has
problems - We have no idea of the mechanisms
- We have no idea of effector tissue
- We have no in vitro model
8Mapping trypanotolerance loci of the NDama
would allow the rapid introgression of desired
traits from other breeds into the NDama, while
retaining the trypanotolerance traits or the
rapid introgression of the trypanotolerance trait
from the NDama to other breeds. Mapping of
trypanotolerance loci would also be the first
step in their eventual cloning and manipulation
through genetic engineering techniques
M. Soller and J.S. Beckmann, FAO Consultation
Report, March 1987
CCER June 2002
9Contribution of 10 genes from Boran and NDama
cattle to reduction in degree of trypanosomosis
Boran (susceptible)
NDama (tolerant)
The NDama and Boran each contribute
trypanotolerance alleles at 5 of the 10 most
significant QTL, indicating that a synthetic
breed could have even higher tolerance than the
NDama.
10In mice, we mapped three genomic regions which
determine survival time following T. congolense
infection
0
D17Mit46
D17Mit16
D5Mit233
D17Mit7
40
D5Mit114
D5Mit24
D1Nds2
MMU17
D1Mit102
80
D1Mit113
D1Mit403
MMU5
MMU1
120cM
11- What are these genes ?
- How do they affect survival ?
- What response pathways are common to
mouse/cow/human ? - What does that tell us about how to survive
trypanosome challenge ? - The mapping data gives us a point of attack for a
functional approach.
12Functional genomics technology allows us to look
at what genes respond to infectionAnd
especially what genes respond differently to
infection
13Expression analysis of cow and mouse, resistant
and susceptible.
14 Expression studiesCow Ndama vs Boran time
course Ndama x Boran backcross Mouse C57
vs A/J Balb/c time course C57A/J
congenics Various mouse mutants (C57A/J BAC
transgenics)
15Microarray design at each time point
Resistant C57BL/6
Susceptible AJ
16Mouse time course. Liver.
17Cholesterol metabolism
18Endogenous cholesterol production increases after
infection
19Total Cholesterol levels
20Congenics
- Lines fixed for alternative alleles of each QTL
on a susceptible background.
21Breeding congenic Mice carrying the
Trypanotolerance QTLs
22(No Transcript)
23(No Transcript)
24(No Transcript)
25(No Transcript)
26(No Transcript)
27(No Transcript)
28Cxcl1 - inflammatory response
29Cattle Microarray Time Course Design
30Cattle Microarray Time Course Design
31PCA Liver day 0. 1st component tissue, this is 2nd
- Principle components analysis of data from
genome-wide expression analysis comparing gene
expression in liver of Ndama (red) vs Boran
(blue) in response to infection with T.
congolense. Light colour day 29 post infection,
dark day 32 post infection. Components 1 and 2.
(Components 3 and 4 separate by day post
infection)
32And the same data for spleen. The biggest effect
we see (after tissue) is breed.
- Principle components analysis of data from
genome-wide expression analysis comparing gene
expression in spleen of Ndama (red) vs Boran
(blue) in response to infection with T.
congolense. Light colour day 29 post infection,
dark day 32 post infection. Components 1 and 2.
(Components 3 and 4 separate by day post
infection)
33Cattle Microarrays
Paraoxonase 3 Chr 4 Near Parasitaemia QTL??
34Paraoxonase 3 (PON3)
- PON1 knockout mice are more susceptible to T.
congolense - PON3 is a 40-kDa protein associated with the high
density lipoprotein fraction of serum - PON3 rapidly hydrolyzes lactones such as statin
prodrugs (e.g. lovastatin) - PON3 is more efficient than rabbit PON1 in
protecting low density lipoprotein from
copper-induced oxidation
35Expression profiling of NDama-Boran backcross
cattle
36African cows to Salford ICU
High cholesterol in African cattle identified as
a protective factor against death from
trypanosomiasis
Is high cholesterol a protective factor in humans
undergoing extreme inflammation?
ICU data and physicians in Salford lab
accessible, plus heamoglobin, creatinine and
glucose clampingfrom normal ICU practice
minimises major confounders
Data cleaning, meta-data capture, analysis
37(No Transcript)
38What to do with candidate genes
- All the obvious things
- Plus
- Exploit the unique populations of cattle
- Recently admixed resistant susceptible
- Multiple resistant breeds
- Multiple resistant species (sheep, goat, wildlife)
39(No Transcript)
40Shirakawa Institute of Animal Genetics
KARI-TRC
41Intersection of Cholesterol and Inflammatory
pathways
Th2 bias
Suppression of cholesterol synthesis
Dunn et al Journal of Experimental Medicine Vol.
203, No. 2, February 20, 2006 401412