Corkins Mark E.

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Corkins
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Mark E.
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  • Article
    Transgenic Xenopus laevis line for in vivo labeling of nephrons within the kidney
    (MDPI AG, 2018-04-06) Corkins, Mark E. ; Hanania, Hannah L. ; Krneta-Stankic, Vanja ; DeLay, Bridget D. ; Pearl, Esther J. ; Lee, Moonsup ; Ji, Hong ; Davidson, Alan J. ; Horb, Marko E. ; Miller, Rachel K.
    Xenopus laevis embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between Xenopus and humans, allowing for the study of human disease-causing genes. Xenopus embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this cdh17:eGFP line, showing green fluorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of cdh17:eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes.
  • Preprint
    Targeted knockout of lhx1 via CRISPR/Cas9 gene editing in the Xenopus laevis kidney
    ( 2017-09-29) DeLay, Bridget D. ; Corkins, Mark E. ; Hanania, Hannah L. ; Salanga, Matthew C. ; Deng, Jian Min ; Sudou, Norihiro ; Taira, Masanori ; Horb, Marko E. ; Miller, Rachel K.
    Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus. The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.