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11 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Mouse Models of Periventricular Leukomalacia
Yan Shen 1, Jennifer M. Plane 1, Wenbin Deng 1
1Department of Cell Biology and Human Anatomy Institute for Pediatric Regenerative Medicine School of, University of California, Davis

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

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Immunology and Infection

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model
Mary Zimmerman *1, Xiaolin Hu *1, Kebin Liu 1
1Department of Biochemistry and Molecular Biology, Medical College of Georgia

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

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Medicine

miRNA Expression Analyses in Prostate Cancer Clinical Tissues
Nathan Bucay 1, Varahram Shahryari 1, Shahana Majid 1, Soichiro Yamamura 1, Yozo Mitsui 1, Z. Laura Tabatabai 1, Kirsten Greene 1, Guoren Deng 1, Rajvir Dahiya 1, Yuichiro Tanaka 1, Sharanjot Saini 1
1Department of Urology, Veterans Affairs Medical Center, San Francisco, University of California San Francisco

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

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Medicine

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis
Amy V. Paschall 1,2,3, Kebin Liu 1,2,3
1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, 2Georgia Cancer Center, Augusta University, 3Charlie Norwood VA Medical Center

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

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Medicine

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer
Varahram Shahryari 1, Hannah Nip 1, Sharanjot Saini 1, Altaf A. Dar 2, Soichiro Yamamura 1, Yozo Mitsui 1, Melissa Colden 1, Nathan Bucay 1, Laura Z. Tabatabai 1, Kirsten Greene 1, Guoren Deng 1, Yuichiro Tanaka 1, Rajvir Dahiya 1, Shahana Majid 1
1Department of Urology, VA Medical Center and UCSF, 2California Pacific Medical Center Research Institute

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

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Developmental Biology

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes
Andrew S. Riching 1, Yuanbiao Zhao 1, Yingqiong Cao 1, Pilar Londono 1, Hongyan Xu 2, Kunhua Song 1
1Division of Cardiology, Department of Medicine, University of Colorado Anschutz Medical Campus, 2Department of Population Health Sciences, Medical College of Georgia, Augusta University

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-β signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

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Genetics

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice
Xuan Su *1,2, Yan Shen *1, Yue Jin *1,2, Meng Jiang 2, Neal Weintraub 1, Yaoliang Tang 1
1Vascular Biology Center, Medical College of Georgia, Augusta University, 2Renji Hospital, School of Medicine, Shanghai Jiaotong University

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

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Developmental Biology

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors
Yue Jin 1, Yan Shen 1, Xuan Su 1, Neal Weintraub 1, Yaoliang Tang 1
1Medical College of Georgia, Augusta University

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

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Cancer Research

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients
Divya Bhagirath 1, Rajvir Dahiya 2, Shahana Majid 2, Z. Laura Tabatabai 3, Sharanjot Saini 1
1Department of Biochemistry and Molecular Biology, Augusta University, 2Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, 3Department of Pathology, Veterans Affairs Medical Center and University of California, San Francisco

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

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Engineering

Glass-Based Devices to Generate Drops and Emulsions
Josefa Guerrero 1, Javier Rojo 2, Alexis de la Cotte 2, Luis Manuel Aguilera-Sáez 3, Enric Vila 3, Alberto Fernandez-Nieves 2,4,5
1Department of Chemistry and Physics, Augusta University, 2Department of Condensed Matter Physics, University of Barcelona, 3Agrobío S.L., 4ICREA - Institució Catalana de Recerca i Estudis Avançats, 5Institute of Complex Systems (UBICS), University of Barcelona

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

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Biology

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells
Ryan Tomaszewski 1,2, Pragya Rajpurohit 3, Mei Cheng 1,2, Amany Tawfik 1,2,3
1Eye Research Institute, Oakland University, 2Eye Research Center (OUWB)/ERC, William Beaumont School of Medicine, 3Oral Biology and Diagnostic Sciences, Dental College of Georgia, Augusta University

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

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