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  Infection-dependent MHC expression in the three-spined stickleback, Gasterosteus aculeatus

Hibbeler, S. (2006). Infection-dependent MHC expression in the three-spined stickleback, Gasterosteus aculeatus. Diploma Thesis, Christian-Albrechts-Universität, Kiel.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-D822-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-D823-1
Genre: Thesis

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 Creators:
Hibbeler, Sascha1, Author              
Milinski, Manfred1, Advisor              
Reusch, Thorsten1, 2, Referee              
Affiliations:
1Department Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_1445634              
2Department Ecophysiology, Max Planck Institute for Limnology, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_976547              

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 Abstract: 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.

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Language(s): eng - English
 Dates: 2006-12
 Publication Status: Accepted / In Press
 Pages: 65 p.
 Publishing info: Kiel : Christian-Albrechts-Universität
 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
 Rev. Method: -
 Identifiers: eDoc: 294089
Other: Dipl/11347
 Degree: Diploma

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