Oct4-Enhanced Green Fluorescent Protein Transgenic Pigs: A New Large Animal Model for Reprogramming Studies

2011 ◽  
Vol 20 (9) ◽  
pp. 1563-1575 ◽  
Author(s):  
Monika Nowak-Imialek ◽  
Wilfried A. Kues ◽  
Bjoern Petersen ◽  
Andrea Lucas-Hahn ◽  
Doris Herrmann ◽  
...  
Keyword(s):  
Animal Model ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Large Animal Model ◽  
Large Animal ◽  
Transgenic Pigs ◽  
Green Fluorescent

27 AN IMPROVED LARGE ANIMAL MODEL FOR THE STUDY OF ADULT STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 143
Author(s):  
S. Simpson ◽  
L. Gonzalez ◽  
J. Chung ◽  
A. Blikslager ◽  
S. Magness ◽  
...  
Keyword(s):  
Stem Cell ◽  
Green Fluorescent Protein ◽  
Somatic Cell ◽  
Fluorescent Protein ◽  
Large Animal Model ◽  
Cell Populations ◽  
Large Animal ◽  
Transcription Activator ◽  
Green Fluorescent ◽  
Stem Cell Populations

Murine models for the study of adult stem cell populations have broadened the understanding of previously uncharacterized stem cell niches. The development of murine reporter lines for the leucine-rich repeat-containing G-protein-coupled receptor-5 (Lgr5) has highlighted the importance of this gene as a stem cell marker in the stomach, intestine, hair follicle, liver, and kidney in mice. These models however have significant limitations in terms of translational applications because of anatomical and physiological differences between humans and mice. In order to overcome these limitations, we have sought to develop a porcine LGR5 reporter line. We report the generation of a porcine stem cell reporter line using the combination of transcription activator-like effector nucleases and somatic cell NT. Transcription activator-like effector nuclease-mediated homologous recombination was used to drive the integration of an internal ribosome entry site green fluorescent protein fusion into the 3′ untranslated region of the LGR5 locus in porcine fetal fibroblast cells. Multiple cell lines were developed and screened for the proper integration event. Upon confirmation of proper integration by genomic DNA sequencing, these lines were used as donors for somatic cell NT. Transfer of the somatic cell NT reconstructed embryos to a surrogate gilt resulted in 3 live births, and the establishment of a founder line of LGR5-green fluorescent protein reporter pigs. We have begun to characterise these lines, having observed fluorescent labelling of putative stem cell populations in the intestinal crypts and hair follicles from these animals. Many of these observations parallel the expression patterns observed in similar murine models. We have confirmed the fluorescent reporter signal by immunohistochemistry using an anti-green fluorescent protein antibody, and are working towards colocalization studies using anti-LGR5 antibodies and RNA in situ hybridization, as well as the characterisation of additional stem cell populations in the pig. The development of this line of transgenic pigs represents significant progress toward the study of adult stem cells, their progenitors, and the stem cell niche, using a large animal model with an anatomy, physiology, and ability to recapitulate human disease that overcomes the current limitations of rodent models. Funding was provided by NIH R21OD019738.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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