Download Microarray Construction: A personal perspective

Development of a Bovine
Macrophage Specific cDNA
Library to Investigate Tropical
Theileriosis
Kirsty Jensen
Tropical Theileriosis
Causative agent:
Vector:
Prevalence:
Economic impact:
Theileria annulata
Hyalomma ticks
200 million cattle at risk
$384.3 million in India/year
Cytospin of T. annulata Infected
Macrophages
Cattle Breeds Exhibiting Differential
Response to T. annulata Infection.
Sahiwal
Bos indicus
Holstein-Friesian
Bos taurus
Tropical Theileriosis
• The breed-specific divergence in disease
progression coincides with the schizont
stage of the parasite.
• Previous work has implicated the infected
macrophage as being central to:– the progression of the disease.
– the breed-specific differences.
Aims
• To investigate the host-pathogen
interactions between bovine macrophages
and T. annulata.
• To elucidate the mechanisms involved in
the breed-specific resistance of cattle to
tropical theileriosis.
Bovine Microarrays
Microarray
Format
Source of Material
BOTL
cDNA
Bovine total leukocyte cDNA library & immune
gene amplicons
Pyxis CattleArray 7600
cDNA
Bovine spleen & placental cDNA libraries
NBFGC
cDNA
MARC & BARC cDNA libraries
BMAM
cDNA
MARC & BARC cDNA libraries
Mammary gland specific
Endometrial/placental
microarray
cDNA
Bovine placental & endometrial cDNA libraries
Bovine immuneendocrine microarray
cDNA
Bovine immune & endocrine gene amplicons
Bovine MHC class I
microarray
oligos
BoLA DRB3 & DQA sequences
RIGUA
oligos
Bovine immune & inflammatory gene amplicons
Affymetrix
oligos
Various cDNA libraries
ARK-Genomics
cDNA
Various cDNA libraries
cDNA Microarray
Source of clones:
Publicly available resources
Collaboration
In house library construction
cDNA Microarray
Construction of cDNA library
Tissue
Tissue status
Normalization v Subtraction
Normalization
Subtraction
RNA
RT-PCR
Denaturation &
Reannealing
Denaturation &
Reannealing
Column
Purification
Column
Purification
Bovine Macrophage cDNA
Library
Normalized
Bos taurus & Bos indicus
Bovine monocytes
Monocytes infected
in vitro with
T. annulata
1, 4, 12, 24, 48 & 96 hour
post infection
Resting
monocytes
Activated monocytes
PMA & Ionomycin
1, 2, 4, 8, 16 & 24 hour
post activation
Bovine macrophage
cDNA library
Activated monocytes
LPS & IFN-
1, 2, 4, 8, 16 & 24 hour
post activation
Established
ex vivo
T. annulata
infected
cell lines
Sequence Analysis
• 10,000 clones were sequenced in
collaboration with the Sanger
Institute
• Initial analysis
– Assessment of library quality:
• Quality of sequences
• Sequence length
• Cluster analysis within the library
Sequence Analysis
• Cluster analysis with EMBL bovine EST
dataset.
• Cluster analysis with EMBL Theileria
annulata EST dataset.
• BLAST analysis with EMBL EST & NR
datasets
• BLAST analysis with human ENSEMBL
dataset.
Composition of the Bovine
Macrophage Library
Unique
45.0%
Bovine
41.2%
RT-PCR Analysis of Unique
Sequences
Monocytes
Activated Infected Ovarian Parasite
+
GAPDH
Tams1
U2
U8
-
+
-
+
-
+
-
RT enzyme
GO Ontologies: Biological Processes
Cancer related
Immune response
Response to stress
Cell adhesion
Cell communication
Cell surface linked signal transduction
Intracellular signaling cascade
Cell organization & biogenesis
Cell growth and/or maintenance
Cell cycle
Cell proliferation
Cell death
Metabolism
Transport
Others
Composition of the bovine macrophage
microarray
Source & Description
Number
Bovine Macrophage Library
ENSEMBL & BLASTN hits
EST hits bovine
human
other
Represent unique clusters
ESTscan hits
Singletons
2372
1076
139
43
234
204
632
MARC1-4/BARC5
312
Amplicons
25
Total
5037
Microarray Experiments
• Experiment 1.
Investigation of the gene expression dynamics of
Holstein & Sahiwal derived macrophages at 2 &
72 hours post T. annulata infection.
– 1) Host-pathogen interactions.
– 2) Breed-specific differences.
• Experiment 2.
Investigation of the gene expression dynamics of
Holstein & Sahiwal derived macrophages at 2 &
16 hours post LPS & IFN- activation.
The BoMP Microarray
Infection Study
• Over 500 genes have been identified that exhibit
differential expression during infection (P<0.05,
fold change >2).
• Over 100 genes have been identified that exhibit
breed-specific differential expression during
infection (P<0.05, fold change >2).
• Over 20 of these genes exhibit breed-specific
differential expression in resting cells (P<0.05,
fold change >2).
Breed-Specific Differential Expression
in Unactivated Monocytes.
4
Fib
2
0
-2
CD9
-4
-6
AOX1
-8
- 10
- 12
Holstein
Sahiwal
NF-kB
AP 373
C1r
Validation of Microarray Results
Holstein
H1 H2 H3 H4 H5 H6
28s
AOX1
NF-kB AP 373
C1r
Fib
CD9
Sahiwal
S1 S2
S3
S4
N
Validation of Microarray Results
30
Fibronectin
CD9
mRNA fold change
25
20
15
10
5
0
H1
H2
H3
Holstein
H4
H5
S1
S2
S3
Sahiwal
S4
S5
Acknowledgements
Roslin Institute
Liz Glass
Mary Clapperton
Susan Craigmile
Edith Paxton
Giles Makins
John Williams
Wilson Lee
David Speed
ARK-Genomics Facility
Richard Talbot
Alison Downing
Frazer Murray
University of Edinburgh
Paul Wright
ENMV, Tunisia
Mohammad Darghouth
Wellcome Trust
Sanger Centre
Ruth Taylor
Jane Rogers
BBSRC