About

Robert Grainger is the W. L. Lyons Brown Professor of Biology at the University of Virginia. His research focuses on eye development and disease, particularly how tissues and organs become directed and committed to their specific fates during embryogenesis. His laboratory extensively studies the formation of the embryonic eye, utilizing the amphibian Xenopus as a model organism due to its accessibility for large-scale embryonic studies and the conservation of eye development processes between frogs and humans. Grainger's work involves the use of molecular and genetic tools, including gene editing techniques, to investigate the genetic basis of eye formation. In addition to his work with Xenopus, Grainger collaborates on research related to human congenital eye diseases, examining how disruptions in genes necessary for normal eye development lead to conditions such as those caused by mutations in PAX6. His lab has developed frog mutants that mirror human eye malformations, providing insights into developmental processes and potential treatments. His research also explores pigmentation in the eye and genetic disorders like Hermansky-Pudlak Syndrome, aiming to understand the underlying mechanisms and improve therapeutic approaches. Grainger's work bridges developmental biology and clinical research, contributing to our understanding of eye formation and associated diseases.

Research topics

• Cell biology
• Genetics
• Biology
• Anatomy
• Neuroscience
• Molecular biology
• Computational biology
• Internal medicine

Selected publications

• Modelling human genetic disorders in <i>Xenopus tropicalis</i>

  Disease Models & Mechanisms · 2024-05-01 · 8 citations

  articleOpen accessSenior author

  Recent progress in human disease genetics is leading to rapid advances in understanding pathobiological mechanisms. However, the sheer number of risk-conveying genetic variants being identified demands in vivo model systems that are amenable to functional analyses at scale. Here we provide a practical guide for using the diploid frog species Xenopus tropicalis to study many genes and variants to uncover conserved mechanisms of pathobiology relevant to human disease. We discuss key considerations in modelling human genetic disorders: genetic architecture, conservation, phenotyping strategy and rigour, as well as more complex topics, such as penetrance, expressivity, sex differences and current challenges in the field. As the patient-driven gene discovery field expands significantly, the cost-effective, rapid and higher throughput nature of Xenopus make it an essential member of the model organism armamentarium for understanding gene function in development and in relation to disease.

  PublisherOA PDFDOI

• Report on the 2021 Aniridia North America symposium on PAX6, aniridia, and beyond

  The Ocular Surface · 2023-05-27 · 10 citations

  review1st authorCorresponding
  PublisherDOI

• <i>Six3</i> acts independently of <i>Pax6</i> to provide an essential contribution to lens development

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-01-10

  preprintOpen accessSenior authorCorresponding

  ABSTRACT The Six3 transcription factor is essential for forebrain and eye development, and SIX3 mutations cause the congenital disorder holoprosencephaly. We created a six3 mutant in Xenopus tropicalis with a mild holoprosencephaly phenotype, and unlike mouse Six3 mutants that are headless/eyeless, the Xenopus mutant forms some eye structures, allowing direct study of Six3 function in eye formation. We focus here on striking deficits in lens formation. Early lens induction occurs normally in the mutant, e.g., the essential eye gene pax6 , is activated in lens ectoderm, persisting in the eye to a late developmental stage, but in many embryos the lens fails to form. We found that bmp4, bmp7 . 1, smad7, dll1, dlc, mab21l1 and/or mab21l2 , previously unknown as six3 eye targets, are downregulated in the mutant. We show that six3 is required for lens formation, acting primarily in developing retina during neurulation through BMP and Notch signaling, and that mab21l1/mab21l2 regulate(s) this BMP activity. This work reveals previously unrecognized essential roles for six3 in eye development, identifying its key role in signaling needed for lens formation, and acting independently of pax6 activity. SUMMARY STATEMENT This study identifies the six3 transcription factor as the mediator of key inductive signals driving lens formation, acting independently of pax6 in early phases of lens formation.

  PublisherOA PDFDOI

• Erratum: Best Practices for<i>Xenopus tropicalis</i>Husbandry

  Cold Spring Harbor Protocols · 2023-07-01

  erratumOpen accessSenior author
  PublisherOA PDFDOI

• Genetics and Gene Editing Methods in<i>Xenopus laevis</i>and<i>Xenopus tropicalis</i>

  Cold Spring Harbor Protocols · 2022 · 4 citations

  Senior authorCorresponding
  • Biology
  • Computational biology
  • Genetics

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  PublisherDOI

• Preparation of Intact Nuclei for Single-Nucleus Omics Using Frozen Cell Suspensions from Mutant Embryos of<i>Xenopus tropicalis</i>

  Cold Spring Harbor Protocols · 2022-08-11 · 3 citations

  articleSenior author

  Single-cell omics such as single-cell RNA-sequencing (RNA-seq) have been used extensively to obtain single-cell genome-wide expression data. This technique can be used to compare mutant and wild-type embryos at predifferentiation stages when individual tissues are not yet formed (therefore requiring genotyping to distinguish among embryos), for example, to determine effects of mutations on developmental trajectories or congenital disease phenotypes. It is, however, hard to use single cells for this technique, because such embryos or cells would need to be frozen until genotyping is complete to capture a given developmental stage precisely, but intact cells cannot be isolated from frozen samples. We developed a protocol in which high-quality nuclei are isolated from frozen cell suspensions, allowing for genotyping individual embryos based on a small fraction of a single embryo suspension. The remaining suspension is frozen. After genotyping is complete, nuclei are isolated from embryo suspensions with the desired genotype and encapsulated in 10× Genomics barcoded gel beads for single-nucleus RNA-seq. We provide a step-by-step protocol that can be used for single transcriptomic analysis as well as single-nucleus chromatin accessibility assays such as ATAC-seq. This technique allows for high-quality high-throughput single-nucleus analysis of gene expression in genotyped embryos. This approach may also be valuable for collection of wild-type embryonic material, for example, when collecting tissue from a particular developmental stage. In addition, freezing of tissue suspensions allows precise staging of collected embryos or tissue that may be difficult to manage when collecting and processing cells from living embryos for single-cell RNA-seq.

  PublisherDOI

• Best Practices for<i>Xenopus tropicalis</i>Husbandry

  Cold Spring Harbor Protocols · 2022-10-25 · 3 citations

  articleOpen accessSenior author

  Xenopus tropicalis has been adopted by laboratories as a developmental genetic system because of its diploid genome and short generation time, contrasting with Xenopus laevis , which is allotetraploid and takes longer to reach sexual maturity. Because X. tropicalis has been introduced more recently to many laboratories, some specific methods more appropriate for handling of eggs and embryos of X. tropicalis are still not widely known to researchers who use X. laevis . Here we highlight some recommendations and opportunities possible with this model system that complement existing X. tropicalis procedures. Of particular importance, because of the value of generating genetically modified lines for researchers using X. tropicalis, we describe a procedure for sterilizing embryos, which could be applied to both species of Xenopus , but might be particularly useful for raising genetically modified animals in X. tropicalis . This protocol will help ensure that a colony will have a high probability of being free of pathogens known to be serious threats to Xenopus health.

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• Gynogenetic Production of Embryos in<i>Xenopus tropicalis</i>Using a Cold Shock Procedure: Rapid Screening Method for Gene Editing Phenotypes

  Cold Spring Harbor Protocols · 2022-08-11 · 4 citations

  articleSenior author

  Gynogenesis is a form of parthenogenesis in which eggs require sperm for fertilization but develop to adulthood without the contribution of paternal genome information, which happens naturally in some species. In Xenopus , gynogenetic diploid animals can be made experimentally. In mutagenesis strategies that only generate one allele of a recessive mutation, as might occur during gene editing, gynogenesis can be used to quickly reveal a recessive phenotype in eggs carrying a recessive mutation, thereby skipping one generation normally required to screen by conventional genetics. Xenopus oocytes do not complete meiosis until shortly after fertilization, and the second polar body is retained in fertilized eggs. Using ultraviolet (UV)-irradiated sperm, fertilization can be triggered without a genetic paternal contribution. Upon applying cold shock at the proper time to such embryos, ejection of the second polar body can be suppressed and both maternal sister chromatids are retained, leading to the development of gynogenetic diploid embryos. Because the genome of the resultant animals consists of recombined sister chromatids because of crossover events during meiosis, it is not completely homozygous throughout the whole genome. Nevertheless, the genome is homozygous at some loci proximal to the centromere that are unlikely to undergo recombination during meiosis and homozygous at reduced frequency if mutations are farther from the centromere, but still generally at a scorable level. Therefore, this technique is useful for rapid screening phenotypes of recessive mutations in such regions. We describe here a step-by-step protocol to achieve cold shock-mediated gynogenesis in Xenopus tropicalis .

  PublisherDOI

• Production of Transgenic F₀Animals and Permanent Lines by Sperm Nuclear Transplantation in<i>Xenopus tropicalis</i>

  Cold Spring Harbor Protocols · 2022-10-25 · 2 citations

  articleSenior author

  Early efforts in the 1980s showed that DNA microinjected into Xenopus embryos could be integrated into the genome and transmitted through the germline at low efficiency. Subsequent studies revealed that transgenic lines, typically with multiple-copy inserts (e.g., to develop bright fluorescent protein-reporter lines), could be created via sperm nuclear injection protocols such as the one entitled restriction enzyme-mediated insertion, or REMI. Here we describe a refined sperm nuclear injection procedure, with a number of alterations, including elimination of a potential DNA-damaging restriction enzyme treatment, aimed at making F 0 transgenic animals and transgenic lines in Xenopus tropicalis . This protocol also uses an oocyte extract rather than the egg extract used in older protocols. These changes simplify and improve the efficiency of the procedure.

  PublisherDOI

• Homology-Directed Repair by CRISPR–Cas9 Mutagenesis in<i>Xenopus</i>Using Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos

  Cold Spring Harbor Protocols · 2022-08-11 · 4 citations

  articleOpen access

  We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing in Xenopus embryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR–Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilized Xenopus eggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F 1 generation. Therefore, we can avoid the more challenging “oocyte host transfer” technique, which is particularly difficult for Xenopus tropicalis , that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5′-end phosphorothioate-modification to obtain higher-efficiency HDR.

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Recent grants

• NIH Grant R01EY005542

  NIH · $637k · 1991

• NIH Grant R03TW001362

  NIH · $110k · 2003

• NIH Grant R01EY010283

  NIH · $1.5M · 2004

• NIH Grant R01EY006675

  NIH · $4.0M · 2006

• NIH Grant R01EY018000

  NIH · $1.3M · 2012

Frequent coauthors

• Roy C. Ogle

  Old Dominion University

  18 shared

• Jennifer L. Keating

  18 shared

• S A Amero

  Center for Scientific Review

  18 shared

• WJ Murdoch

  University of Wyoming

  18 shared

• Vincent Montoya

  18 shared

• Margaret S. Saha

  William & Mary

  12 shared

• Marilyn Fisher

  Albany Medical Center Hospital

  9 shared

• Jonathan J. Henry

  8 shared