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ID: 294089.0, MPI für Evolutionsbiologie / Evolutionary ecology
Infection-dependent MHC expression in the three-spined stickleback, Gasterosteus aculeatus
Advisors:Milinski, Manfred
Authors:Hibbeler, Sascha
Referees:Reusch, Thorsten
Language:English
Date of Approval (YYYY-MM-DD):2006-12
Type of Thesis (e.g.Diploma):diplom
Name of University:Christian-Albrechts-Universität
Place of University:Kiel
Physical Description 
(e.g. Total Number of Pages):
65 p.
Audience:Not Specified
Table of Contents:1. SUMMARY .................................................................. 5
2. DEUTSCHE ZUSAMMENFASSUNG................................ 7
3. INTRODUCTION......................................................... 9
3.1. MAJOR HISTOCOMPATIBILITY COMPLEX................................................9
3.2. REAL-TIME PCR..................................................................................10
3.3. THESIS OUTLINE ..................................................................................11
4. MATERIAL AND METHODS........................................ 15
4.1. FISH FOR THE INFECTION EXPERIMENT ................................................15
4.2. TREATMENT OF CELL CULTURES .........................................................17
4.3. PRIMER DESIGN ...................................................................................19
4.4. SEQUENCING OF GENES .......................................................................20
4.5. RNA EXTRACTION AND REVERSE TRANSCRIPTION ..............................22
4.6. REAL-TIME PCR..................................................................................23
4.7. HOUSEKEEPING GENES ........................................................................24
4.8. DATA ANALYSIS AND STATISTICAL METHODS .....................................26
5. RESULTS .................................................................. 27
5.1. EVALUATION OF PCR CONDITIONS AND PRIMERS ...............................27
5.2. SELECTION OF HOUSEKEEPING GENES IN CELL CULTURES ...................29
5.2.1. Cultures of head kidney cells ...........................................................29
5.2.2. Cultures of spleen cells ....................................................................31
5.3. SELECTION OF HOUSEKEEPING GENES IN FISH......................................33
5.3.1. Expression in head kidneys ..............................................................33
5.3.2. Expression in spleens .......................................................................34
5.3.3. Expression in gills ............................................................................36
5.4. CELL CULTURES ..................................................................................38
5.4.1. MHC II-expression in cell cultures ..................................................38
5.4.2. Number of cells ................................................................................40
5.5 INFECTION EXPERIMENT......................................................................41
5.5.1. Immune response genes in spleens...................................................41
5.5.2 Immune response genes in head kidneys...........................................44
5.5.3 Immune response genes in gills.........................................................46
6. DISCUSSION............................................................ 49
6.1. PRIMER DESIGN AND PCR ...................................................................49
6.2. HOUSEKEEPING GENE..........................................................................51
6.3. CELL CULTURE EXPERIMENT ...............................................................53
6.4. INFECTION EXPERIMENT......................................................................56
7. LITERATURE ............................................................ 59
8. DANKSAGUNG.......................................................... 63
STELLUNGNAHME ........................................................ 65
Abstract / Description:The study focused on two main topics. On the one hand
primers and a PCR protocol were developed to find a suitable
housekeeping gene for quantitative real-time PCR. On the
other hand this study explored the expression of genes related
to an immune response in cell cultures and organs of living
fish. The main focus lay on the genes of the major
histocompatibility complex (MHC).
The MHC has been studied for several years. This is mainly
because of the central role of MHC molecules in the adaptive
immune response and the high number of different alleles in
vertebrate populations. Recent research suggests that – in
addition to the variation in sequences of the genetic code –
genetically based variations in expression of MHC genes do
play a crucial role in evolutionary change. Up to now it has
been shown that expression is heritable, i.e. expression has a
genetic basis in three-spined stickleback (Gasterosteus
aculeatus). Furthermore a negative correlation, which suggests
a compensatory regulation of MHC genes, between expression
levels and number of different MHC alleles has been shown. A
positive correlation between MHC expression and parasite load
has been shown as well.
To date, research has focused on beta-actin as a housekeeping
gene in expression analysis on Gasterosteus aculeatus. Recent
research in other taxa suggests that beta-actin is not the most
stable gene depending on the tissue and the taxa of the
survey.
In the study primers were developed in order to measure gene
expression of candidate housekeeping genes and the immune
response genes. Therefore sequences from the EST-library and
the whole genome shotgun library of the stickleback have been
used to find exon-intron boundaries. The mRNA has been
amplified in a PCR reaction and the product has been
sequenced to check the identity of the PCR product.
Ten candidate genes including beta-actin were tested for stable
gene expression in three different tissues and under several
treatments. The data showed that beta-actin is a good
housekeeping gene in most of the tissues and treatments, but
other genes proved to be more stably expressed. As it was one
of the most stable genes in all examined tissues, especially the
L13a ribosomal binding protein seems to be a good candidate
to replace beta-actin as a general housekeeping gene for
expression analysis.
In another experiment the expression of MHC genes was
stimulated in cell cultures with lysates of two widespread
parasites of the three-spined stickleback, i.e. Diplostomum
pseudospathacaeum and Camallanus lacustris. In order to
enhance comparability, cells from the same organ have been
infected in these cultures. Therefore RNA-Extraction and realtime
PCR were improved in a way that allowed us to measure
gene expression and controls in 15 different PCR runs from
only 105 cells. While MHC expression was higher in cells of
formerly infected fish, no effect of cell treatment could have
been shown. However the cell’s survival rate was higher in
cultures that were infected with parasite lysate.
Furthermore gene expression was measured at five times after
being infected with Diplostomum pseudospathacaeum to
explore the expression of genes correlated to immune response during the first eleven days.
Document Type:Thesis
Affiliations:MPI für Limnologie/Abt. Evolutionsökologie
Identifiers:LOCALID:Dipl/11347 [Signatur/Zugangsnummer]
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