Towards the analyses of cell lineages using conditional gene alterations

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Abstract

The ability to precisely modify the mouse genome is an invaluable tool for any researcher. If an artificial epitope sequence is integrated into target loci in specific cell types, it is possible to generate mice with these cells specifically tagged with the epitope, which can be used for many subsequent studies. Homologous recombination and the Cre/loxP system have been used to generate targeted and conditional transgenic mice, which have provided the basis for many studies into gene function. However, in recent years, improvements in technology have led to the development of RNA and protein based methods of specifically editing DNA sequences at user-selected loci. This thesis aimed to investigate the effectiveness of the novel gene targeting methods TALEN and CRISPR/Cas9. It also aimed to utilize different strains of mice generated using the Cre/loxP system in Trichuris muris, an animal infection model of the human disease Trichuris trichiura. TALENs use a pair of protein-based monomers specific to the sense and anti-sense strand of a target DNA sequence to dimerise a FokI nuclease and initiate a deletion in the genome. As a study into the practical use of this emerging technology TALENs were generated to target Oct-4 (a stem cell marker) in order to integrate an artificial epitope sequence, which could be used for enrichment experiments. The CRISPR/Cas9 is a very efficient RNA-based system used for modifying a target sequence. This system has been utilized to integrate an epitope sequence into the Rosa26 locus, downstream of a floxed STOP codon. This allows for expression of the epitope following the introduction of tissue restricted Cre recombinase.IL-1 is an important cytokine in the immune response towards T.muris. IL-1R1 was conditionally removed in CD4 cells and the role of IL-1 signaling in developing Th1, Th2 and Th17 responses was then studied. Interestingly, IL-1R1fl/fl CD4Cre mice could generate Th1 and Th2 response but showed a reduction in IL-22 and IL-17 production, two key Th17 cytokines. Infected IL-1R1fl/fl CD4Cre mice also displayed increased gut morphology and goblet cell hyperplasia. Therefore, it was concluded that IL-1 signaling from CD4 cells has an important role in host defense and the development of a full Th17 response. It was also shown that removing IL-1R1 in naïve mice had no affect on lymphocyte development.IL-10 is an anti-inflammatory cytokine expressed by gut macrophages, which contributes to homeostatic control of the immune system. IL-10R was specifically removed in the macrophage specific Cre lines LysMCre and also in CX3CR1Cre as a way of comparing the two Cre drivers. The mice were then infected with T.muris and displayed significant inflammation and also failure to expel the worms in the LysMCre model. This suggests a role for this model in future studies of gut macrophages.Clearly, animal models are very important in the study of gene function and also as a method of assessing the application of new technologies such as CRISPR/Cas9. Future work with the artificial epitope specifically targeted into important cell lines will form the basis of many important studies directly applicable to human disorders. As the technologies improve, the scope for developing therapeutics increases and genetic modification has an immeasurable role to play.

Keyword(s)

Artificial Epitope; CD4; CRISPR; Gene Targeting; IL-1; Rosa26; TALEN

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