Editing the genome of chicken primordial germ cells to introduce alleles and study gene function

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are develope

Enzymatically Catalyzed Degradation of Biodegradable Polymers Investigated by Means of a Semiconductor-based Field-effect Sensor

A semiconductor field-effect device has been used for an enzymatically catalyzed degradation of biopolymers for the first time. This novel technique is capable to monitor the degradation process of multiple samples in situ and in real-time. As model system, the degradation of the biopolymer poly(D, L-lactic acid) has been monitored in the degradation medium containing the enzyme lipase from Rhizomucor miehei. The obtained results demonstrate the potential of capacitive field-effect sensors for degradation studies of biodegradable polymer

Sensor System for in-situ and Real-time Monitoring of Polymer (bio)degradation

A sensor system for investigating (bio)degradationprocesses of polymers is presented. The system utilizes semiconductor field-effect sensors and is capable of monitoring the degradation process in-situ and in real-time. The degradation of the polymer poly(d,l-lactic acid) is exemplarily monitored in solutions with different pH value, pH-buffer solution containing the model enzyme lipase from Rhizomucormiehei and cell-culture medium containing supernatants from stimulated and non-stimulated THP-1-derived macrophages mimicking activation of the immune system

High-energy milled, Eu3 +-doped fresnoite glass-ceramic powders: Structural characterization and luminescent properties

Fresnoite glass-ceramic powders were investigated as luminescent materials, due to their pronounced blue luminescence upon UV excitation. However, the luminescence intensity of mechanically milled luminescent materials decreases by reducing the particle size, because of the induced surface damage of the particles. Thus, the preparation of luminescent nano powders still represents a challenge in the processing of luminescent materials. In this work, different milling routes were carried out in order to achieve a submicron Eu3 +-doped Fresnoite (Ba2TiSi2O8) glass-ceramic powder, keeping sufficient luminescence intensity. The milling parameters studied were: milling medium, milling balls-to-powder mass ratio, rotation speed and time of milling. The microstructure of the powders was evaluated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The luminescence emission at UV excitation was recorded and related with the microstructure observed. The local environment of the Eu3 + ions changes with the particle size, as shown from the red-to-orange emission ratio. An optimized milling route is proposed, which enables to produce particles as small as possible with the highest possible emission intensity