36 research outputs found
Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation
Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model syste
Quantitative analysis of the tomato nuclear proteome during <i>Phytophthora capsici </i>infection unveils regulators of immunity
Finishing the euchromatic sequence of the human genome
The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead
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
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Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9.
Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa
Simultaneous reprogramming and gene editing of human fibroblasts
The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6–8 weeks), and cost-effective
Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9
Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa
Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts
SummaryThe derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes