Dr Don Love
Dr Don Love
Dr Don Love
https://unidirectory.auckland.ac.nz/profile/d-loveHonorary, Academic The University of Auckland: Biological Sciences
Research | Current
- Functional Genomics
Functional genomics involves the development and application of global experimental approaches to assess gene function by making use of the information and reagents provided by genome sequencing and mapping. In this context, we have embarked on a dual strategy for effecting changes in gene expression in the zebrafish in order to assess the consequences of these changes at a global level.
These strategies involve targeted mutagenesis as well as gene down-regulation to model a variety of human disorders, with an emphasis on Duchenne muscular dystrophy (DMD). This neuromuscular disorder is due to mutations in the muscle protein, dystrophin, which cause loss of dystrophin at the sarcolemma and destabilisation of a multi-protein dystrophin-associated glycoprotein complex. We propose that modelling in the zebrafish offers a better means of assessing the real-time dynamics of gene expression and protein localisation during muscle development compared to mouse studies.
Our experimental approach is essentially two-fold. First, to undertake the selective mutagenesis of the dystrophin gene in the developing zebrafish by introducing peptide nucleic acids (PNAs) that will target this gene. Secondly, to use RNA interference to effect transient down-regulation of the zebrafish dystrophin gene. Each of these strategies is presented below.
Gene targeting reagents: Triplex-forming oligonucleotides, side-by-side minor groove binders, and helix-invading peptide nucleic acids (PNAs) are effective as gene-specific transcriptional blocking agents. The latter reagent is stable against nucleases and proteases, binds independently of salt concentration and, due to its neutral backbone, has a much higher affinity for nucleic acids than do DNA/DNA duplexes. In addition, modified PNAs offer the possibility of not only blocking transcription, but of introducing mutations in the targeted region during DNA replication. Several groups have reported introducing mutations in mammalian cells and yeast using psoralen-conjugated oligonucleotides. Psoralen is a bifunctional photoreagent that introduces a covalent crosslink into target sequences following irradiation at 365nm. We are currently using a number of modified PNAs, including psoralen-conjugated PNAs, as part of a collaborative project that addresses the targeting of zebrafish genes.
Translational knock-down: RNA interference (RNAi), which involves the use of double-stranded (ds) RNA, has been successfully applied to knockdown the expression of specific genes in plants, D. melanogaster, C. elegans, trypanosomes, planaria, hydra, and several vertebrate species including the mouse and zebrafish. As a functional genomic tool, RNAi has great potential for the functional analysis of uncharacterised genes and has already been widely applied to high throughput screens in C. elegans and has recently been shown to be effective in cultured mammalian cells in the form of short interfering RNAs.
Several studies have reported non-specific effects following the direct introduction of dsRNA into zebrafish. However, these effects may simply be due to the injection of large quantities of dsRNA into the developing embryo during a period of precisely controlled expression of a vast array of vertebrate developmental genes. In this respect, neither the use of dsRNA-producing vectors, nor an examination of the effects of RNAi on global gene expression, has been undertaken in the zebrafish. Our current research is directed to addressing these two concerns.
An ancillary project that falls within the area of functional genomics is the introduction of exogenous genes into zebrafish embryos. Our focus here is the modelling of Huntington Disease in the zebrafish. We have constructed a variety of recombinants that carry expanded polyglutamine repeats under the control of a constitutive promoter as well as the zebrafish HD gene promoter. The effect of expression of these recombinants on endogenous gene expression in the zebrafish will be examined using microarrays.
- Chemical Genomics
The identification of new medicines and the development of therapeutics involve the investigation and exploration of defined molecule interactions with complex biological processes. This specificity of action can provide drug prototypes and involves the modulation of gene product function, which comprises the area of Chemical Genomics. This field has been enhanced recently by studying the effects of drug prototypes on not only pure protein targets, but also on an organism's global network of protein interactions.
This type of analysis has involved the development of whole-organism gene expression microarrays in which an organism's entire protein-coding potential (expressed sequences) is spotted onto glass slides in high-density arrays. The slides are subsequently hybridised using fluorescently labelled reverse-transcribed RNA and the slides scanned to detect fluorescent signal. The analysis of these signals provides a measure of expression levels of the genes that are represented on the array. To date, this type of work has been undertaken using invertebrate species only, but has overlooked the use of zebrafish as a model of vertebrate development.
In terms of those compounds that have been studied in the context of chemical genomics, a range of novel and structurally diverse natural products has been isolated from marine organisms (Ascidians and Sponges) collected from shallow water sites around the coast of New Zealand. Such metabolites include terpenes, sulfur-containing alkaloids, purine derivatives and amino-acid derived compounds. Many marine natural products exhibit wide ranges of pharmaceutically interesting biological activities, such as antitumour, antiviral and antimicrobial properties. However, some natural products lack activity in these assays, which are limited in scope and fail to dissect the effect of compounds on complex biological processes.
We are interested in these natural products as they may collectively hold the key to providing valuable information about cellular function and development while offering a new biological resource for medicines and therapeutics.
- Molecular-Based diagnostics
The molecular analysis of the neurodegenerative disorder Adrenoleukodystrophy (ALD) has been undertaken due to the availability of a large New Zealand pedigree manifesting this X-linked disease. The sequencing of ectopic transcripts from lymphoblasts of affected individuals led to the identification of the disease-causing mutation in the ALD gene in this family. A collaborative project has been established with Dr Martin Kennedy of the Christchurch School of Medicine in order to develop a mouse model for ALD.
A DNA-based assay for Huntington disease (HD) has been developed to provide a rapid test for predicting individuals at-risk of HD. This assay was passed on to the molecular genetics laboratory of Auckland Hospital in order to provide a national service for DNA-based HD diagnostics. At the research level the assay has been modified to give a fluorescently labelled PCR product. This modification has enabled the sizing of amplified products using an Applied Biosystems Model 373/377/310 DNA sequencer. The assay has been applied to an examination of somatic cell variation in the (CAG) repeat of the HD locus in those individuals who have developed the disease. Particular emphasis has been placed on the analysis of the HD locus in brain cryosections taken from post-mortem material of affected individuals. Our studies have shown a correlation between those areas of the brain exhibiting somatic variation and those exhibiting apoptosis.
Collaborative research has been undertaken with Associate Professor John French of the Cardiology Unit (Greenlane Hospital) in the mutation screening of genes implicated in familial hypertrophic cardiomyopathy and X-linked dilated cardiomyopathy.
Recent work, in collaboration with Dr Madhuri Hegde of Baylor College of Medicine, has involved the development of improved methods to screen for mutations in the APC gene implicated in familial adenomatous polyposis (FAP). This research has involved the use of DHPLC analysis, which is also being used to screen for mutations in many of the genes implicated in the genetically heterogeneous neuromuscular disorder, limb girdle muscular dystrophy (LGMD). The latter protocol arose from our earlier studies concerned with the screening of the BRCA1 and BRCA2 genes in patients with a family history of breast and ovarian cancer.
The School of Biological Sciences has invested in a Stanford-type microarray platform for the arraying of cDNAs and oligonucleotides. An Axon 4000B GenePix scanner and associated software support the arrayer in our attempts to screen for the effects of perturbed gene expression in the zebrafish brought about by DNA-based as well as chemical reagents.
Areas of expertise
Molecular, Cellular & Developmental Biology
Selected publications and creative works (Research Outputs)
- Marcondes, L., Crawford, J., Earle, N., Smith, W., Hayes, I., Morrow, P., ... Skinner, J. R. (2018). Long QT molecular autopsy in sudden unexplained death in the young (1-40 years old): Lessons learnt from an eight year experience in New Zealand. PloS one, 13 (4)10.1371/journal.pone.0196078
- Whitford, W., Hawkins, I., Glamuzina, E., Wilson, F., Marshall, A., Ashton, F., ... Lehnert, K. (2017). Compound heterozygous SLC19A3 mutations further refine the critical promoter region for biotin-thiamine-responsive basal ganglia disease. Cold Spring Harbor Molecular Case Studies, 3 (6)10.1101/mcs.a001909
Other University of Auckland co-authors: Jessie Jacobsen, Klaus Lehnert, Russell Snell, Whitney Whitford
- Leong, I. U. S., Stuckey, A., Belluoccio, D., Fan, V., Skinner, J. R., Prosser, D. O., & Love, D. R. (2017). Massively Parallel Sequencing of Genes Implicated in Heritable Cardiac Disorders: A Strategy for a Small Diagnostic Laboratory. Medical sciences (Basel, Switzerland), 5 (4).10.3390/medsci5040022
Other University of Auckland co-authors: Vicky Fan
- McKeage, M., Elwood, M., Tin Tin, S., Khwaounjoo, P., Aye, P., Li, A., ... Kingston, N. (2017). EGFR mutation testing of non-squamous NSCLC: Impact and uptake during implementation of testing guidelines in a population-based registry cohort from northern New Zealand. Targeted Oncology, 12 (5), 663-675. 10.1007/s11523-017-0515-4
Other University of Auckland co-authors: Sandar Tin Tin, Mark McKeage, Mark Elwood, Phyu Aye
- Shepherd, P., Sheath, K. L., Tin Tin, S., Khwaounjoo, P., Aye, P. S., Li, A., ... Elwood, J. M. (2017). Lung cancer mutation testing: A clinical retesting study of agreement between a real-time PCR and a mass spectrometry test. Oncotarget, 8 (60), 101437-101451. 10.18632/oncotarget.21023
Other University of Auckland co-authors: Mark McKeage, Sandar Tin Tin, Mark Elwood, Phyu Aye
- Lahrouchi, N., Raju, H., Lodder, E. M., Papatheodorou, E., Ware, J. S., Papadakis, M., ... Crawford, J. (2017). Utility of Post-Mortem Genetic Testing in Cases of Sudden Arrhythmic Death Syndrome. Journal of the American College of Cardiology, 69 (17), 2134-2145. 10.1016/j.jacc.2017.02.046
- Mc Cormack, A., Claxton, K., Ashton, F., Asquith, P., Atack, E., Mazzaschi, R., ... Sheath, K. (2016). Microarray testing in clinical diagnosis: an analysis of 5,300 New Zealand patients. Molecular Cytogenetics, 9 (1).10.1186/s13039-016-0237-9
- McKeage, M. J., Elwood, J. M., Tin, S. T., Khwaounjoo, P., Aye, P., Li, A., ... Lewis, C. (1/12/2016). Population-level impact of EGFR mutation testing in non-squamous NSCLC. ANNALS OF ONCOLOGY.
Other University of Auckland co-authors: Mark McKeage, Mark Elwood