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The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts
The sites of nascent DNA synthesis were compared
with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells
were compared with the distribution of PCNA by
indirect immunofluorescence microscopy and laser
scanning confocal microscopy (LSCM). In common
with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe
coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly,
nuclei which had been isolated from quiescent
cultures of HDF were innoculated into cell-free
extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated
at multiple discrete spots some 15min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.Brunel Open Access Publishing Fun
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