Publications by authors named "Thai Binh Pham"

8 Publications

  • Page 1 of 1

Microinjection Method for Anopheles gambiae Embryos.

J Vis Exp 2021 Jul 7(173). Epub 2021 Jul 7.

Department of Microbiology & Molecular Genetics, University of California, Irvine; Department of Molecular Biology & Biochemistry, University of California, Irvine;

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.
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http://dx.doi.org/10.3791/62591DOI Listing
July 2021

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.

J Vis Exp 2021 Jun 28(172). Epub 2021 Jun 28.

Department of Microbiology & Molecular Genetics, University of California, Irvine; Department of Molecular Biology & Biochemistry, University of California, Irvine;

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.
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http://dx.doi.org/10.3791/62607DOI Listing
June 2021

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes.

J Vis Exp 2021 05 1(171). Epub 2021 May 1.

Department of Microbiology & Molecular Genetics, University of California, Irvine; Department of Molecular Biology & Biochemistry, University of California, Irvine;

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.
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http://dx.doi.org/10.3791/62588DOI Listing
May 2021

Next-generation gene drive for population modification of the malaria vector mosquito, .

Proc Natl Acad Sci U S A 2020 09 24;117(37):22805-22814. Epub 2020 Aug 24.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA 92697-4025;

A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, The drive system targets the gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro, with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.
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http://dx.doi.org/10.1073/pnas.2010214117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502704PMC
September 2020

Global Governing Bodies: A Pathway for Gene Drive Governance for Vector Mosquito Control.

Am J Trop Med Hyg 2020 09;103(3):976-985

Department of Microbiology and Molecular Genetics, University of California, Irvine, California.

Gene drive technologies represent powerful tools to develop vector control strategies that will complement the current approaches to mitigate arthropod-borne infectious diseases. The characteristics of gene drive technologies have raised additional concerns to those for standard genetically engineered organisms. This generates a need for adaptive governance that has not been met yet because of the rapid rate of progress in gene drive research. For the eventual release of gene drive insects into wild populations, an international governance network would be helpful in guiding scientists, stakeholders, public opinion, and affected communities in its use. We examined the current institutions and governing bodies among various continents that could have an impact on gene drive governance or the potential to adapt to its future use. Possible governance strategies also are proposed that seek to bridge gaps and promote an ethically sound policy framework. Ideally, governance strategies should be developed before or at the same pace as gene drive research to anticipate field releases and maximize their impact as a public health tool. However, this is not likely to happen as it takes years to develop global accords, and some countries may choose to move ahead independently on the new technology.
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http://dx.doi.org/10.4269/ajtmh.19-0941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470596PMC
September 2020

Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species .

Biotechniques 2020 04 10;68(4):172-179. Epub 2020 Feb 10.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA 92697-4025, USA.

CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito : Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes.
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http://dx.doi.org/10.2144/btn-2019-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177198PMC
April 2020

Experimental population modification of the malaria vector mosquito, Anopheles stephensi.

PLoS Genet 2019 12 19;15(12):e1008440. Epub 2019 Dec 19.

Department of Microbiology & Molecular Genetics, University of California, Irvine, California, United States of America.

Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (≤ 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.
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http://dx.doi.org/10.1371/journal.pgen.1008440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922335PMC
December 2019

Kinetics of dengue non-structural protein 1 antigen and IgM and IgA antibodies in capillary blood samples from confirmed dengue patients.

Am J Trop Med Hyg 2014 Mar 27;90(3):438-43. Epub 2014 Jan 27.

Laboratoire de Virologie, Centre National de Référence des Arbovirus, Laboratoire Associé, Région Antilles Guyane, Institut Pasteur de la Guyane, Cayenne, French Guiana; Far East Medical Vietnam Limited, Ho Chi Minh City, Vietnam; Pasteur Institute of Ho Chi Minh City, Ho Chi Minh City, Vietnam; Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, Cayenne, French Guiana; Centre d'Epidémiologie et de Santé Publique des Armées, Marseille, France; Unité Mixte de Recherche Scientifique 872, Pôle 4, Equipe 16, Institut National de la Santé et de la Recherche Médicale, Centre de Recherches Biomédicales des Cordeliers, Paris, France.

Large-scale epidemiological surveillance of dengue in the field and dengue patient management require simple methods for sample collection, storage, and transportation as well as effective diagnostic tools. We evaluated the kinetics of three biological markers of dengue infection-non-structural protein 1 (NS1) antigen, immunoglobulin M (IgM), and IgA-in sequential capillary blood samples collected from fingertips of confirmed dengue patients. The overall sensitivities and specificities of the tests were 96% and 100%, respectively, for NS1, 58.1% and 100%, respectively, for IgM, and 33% and 100%, respectively, for IgA. During the acute phase of the disease, NS1 was the best marker of dengue infection, with a sensitivity of 98.7%, whereas from day 5, all three markers exhibited relevant levels of sensitivity. This first descriptive study of the kinetics of biological markers of dengue in capillary blood samples confirms the usefulness of this biological compartment for dengue diagnosis and argues for its exploitation in community-level and remote settings.
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http://dx.doi.org/10.4269/ajtmh.13-0458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945688PMC
March 2014
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