The pinnacle of four decades of research, induced pluripotent stem cells (iPSCs), and genome edit... more The pinnacle of four decades of research, induced pluripotent stem cells (iPSCs), and genome editing with the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR) now promise to take drug development and regenerative medicine to new levels and to enable the interrogation of disease mechanisms with a hitherto unimaginable level of model fidelity. Autumn 2014 witnessed the first patient receiving iPSCs differentiated into retinal pigmented ep-ithelium to treat macular degeneration. Technologies such as 3D bioprinting may now exploit these advances to manufacture organs in a dish. As enticing as these prospects are, these technologies demand a deeper understanding, which will lead to improvements in their safety and efficacy. For example, precise and more efficient reprogramming for iPSC production is a requisite for wider clinical adoption. Improving awareness of the roles of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) and genomic epigenetic status will contribute to the achievement of these aims. Similarly, increased efficiency, avoidance of off-target effects, and expansion of available target sequences are critical to the uptake of genome editing technology. In this review, we survey the historical development of genetic manipulation and stem cells. We explore the potential of genetic manipulation of iPSCs for in vitro disease modeling, generation of new animal models, and clinical applicability. We highlight the aspects that define CRISPR-Cas as a breakthrough technology, look at gene correction , and consider some important ethical and societal implications of this approach.
The application of transcription factor activated luciferase reporter cassettes in vitro is wides... more The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables noninvasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation
of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice
or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify
LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter
constructs in somatotransgenic mice, respectively.
The pinnacle of four decades of research, induced pluripotent stem cells (iPSCs), and genome edit... more The pinnacle of four decades of research, induced pluripotent stem cells (iPSCs), and genome editing with the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR) now promise to take drug development and regenerative medicine to new levels and to enable the interrogation of disease mechanisms with a hitherto unimaginable level of model fidelity. Autumn 2014 witnessed the first patient receiving iPSCs differentiated into retinal pigmented ep-ithelium to treat macular degeneration. Technologies such as 3D bioprinting may now exploit these advances to manufacture organs in a dish. As enticing as these prospects are, these technologies demand a deeper understanding, which will lead to improvements in their safety and efficacy. For example, precise and more efficient reprogramming for iPSC production is a requisite for wider clinical adoption. Improving awareness of the roles of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) and genomic epigenetic status will contribute to the achievement of these aims. Similarly, increased efficiency, avoidance of off-target effects, and expansion of available target sequences are critical to the uptake of genome editing technology. In this review, we survey the historical development of genetic manipulation and stem cells. We explore the potential of genetic manipulation of iPSCs for in vitro disease modeling, generation of new animal models, and clinical applicability. We highlight the aspects that define CRISPR-Cas as a breakthrough technology, look at gene correction , and consider some important ethical and societal implications of this approach.
The application of transcription factor activated luciferase reporter cassettes in vitro is wides... more The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables noninvasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation
of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice
or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify
LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter
constructs in somatotransgenic mice, respectively.
Uploads
Papers by R. Karda
of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice
or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify
LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter
constructs in somatotransgenic mice, respectively.
of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice
or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify
LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter
constructs in somatotransgenic mice, respectively.