BLT Stages

Abstract Bachelor Project FBT 2018-2019: Generating knockout zebrafish (Danio rerio) lines for modeling human immunity

The Mycobacterium tuberculosis causes tuberculosis in humans. Tuberculosis is still present and is very contagious. The objective of this research is to generate knockout zebrafish (Danio rerio) lines for modeling the human immunity on tuberculosis. This is done by genotyping the zebrafish and breed with them until homozygote mutant lines are formed. This research helps in the search for a new valid working vaccine and creates new insights on the immunity on tuberculosis. The process and methodology for genotyping the larvae and adult zebrafish is already optimized. F0 zebrafish are previously edited with CRISPR-Cas9 that targets the Pycard gene. From the F0 Pycard zebrafish, F1 Pycard zebrafish where generated, genotyped and put into breeding groups. Before being able to start infection experiments and analyze the results, the F2 adult zebrafish needs to be genotyped. Tailfin cuts and lysis are performed on 52 adult Pycard F2 zebrafish, divided into two batches. The DNA is isolated by using ethanol precipitation. After, the DNA is amplified with PCR. The amplification is checked with a 2 % agarose gel. Further, restriction fragment length polymorphism is used to check the genotype of the adult zebrafish. 13 wild type, 23 heterozygote and 16 homozygote Pycard zebrafish were found. The adult zebrafish with the same genotype are then put into the same tanks and breeding groups are created. Human immunity was modeled by infecting F3 homozygote Pycard zebrafish embryos and AB zebrafish as control with Mycobacterium marinum. Their survival was followed until 7 days post fertilization (dpf). After 5 dpf, a difference could be seen between the homozygote and wild type Pycard zebrafish. The homozygote Pycard zebrafish seems to survive the infection better than the WT siblings. In contrast, the AB zebrafish seems to survive better. Based on our results, it would seem that this target gene has an influence on the survival of the infection and could possibly impair the survival from the infection. If the infection experiments are repeated for the different target genes, and the same results are gained, the next step is testing the setting in the mouse (mus musculus) model. If an interesting gene affecting the survival is discovered, it could be used in the development of new medicine and drugs against Mycobacterium infections in humans.

 

Tuberculosis is a bacterial infection that is caused by Mycobacterium tuberculosis and is often found in human lungs. For this research, it is more feasible to use Mycobacterium marinum, a zebrafish pathogen that is less contagious than M. tuberculosis. With this experiment we want to optimize the production of knockout strains of Mycobacterium marinum and the identification of true knockout strains among the colonies. Electroporation of a plasmid that is unable to replicate in mycobacteria is used to construct possible knockout strains of the mycobacteria via homologous recombination consisting of two subsequent cross-over events. As a result, the gene-of-interest will be replaced by a hygromycin resistance gene. The plasmid also contains other marker genes (LacZ and sucrose sensitivity), these genes can be used for colony PCR and sucrose test of potential single- and double-crossover strains. During the optimisation of the PCR protocol, we observed small facts to look at: the use of the correct positive control (pTEC27 versus maxiprep plasmid), time of the growth of the bacteria and the “age” of the culture or the colonies on the plate of the mycobacteria. Secondly, the sucrose test is a suitable way to test for the sensitivity of single crossover bacteria for sucrose. In addition to the optimization, possible knockouts were created (MMAR_0514 and MMAR_4524). The obtained results of the marker gene tests are looking very promising. Since the results of the sucrose test, we know that the chance is bigger that the strains (MMAR_0514 and MMAR_4524) are a single-crossover. Furthermore, the possible knockout strains to confirm the insertion site of the hygromycin resistance gene, in the bacteria. After this, the created knockouts could be tested for infectivity and ability to reactivate by injecting them into the zebrafish.

 

 

Samenvatting eindwerk 2014-2015: Integrated analysis of miRNA and mRNA microarray data in prostate cancer cells