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10 results on '"Thom Saunders"'

1. Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes

Author

Bangmei Wang, Kunyu Li, Amy Wang, Michelle Reiser, Thom Saunders, Richard F Lockey, and Jia-Wang Wang

Subjects
CRISPR cleavage, double-strand break (DSB), homology-directed-repair (HDR), embryonic stem (ES) cells, zygote, Biology (General), QH301-705.5
Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Published
2015
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7. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors

Author

Linda C. Samuelson, Jurgen Schnermann, Josie P. Briggs, Tianxin Yang, Alex Paliege, Thom Saunders, Daqing Sun, and Yuning Huang

Subjects
Male, medicine.medical_specialty, Adenosine, Genotype, Nephron, Biology, Sodium Chloride, Feedback, Renal Circulation, Electrolytes, Mice, Oxygen Consumption, Internal medicine, medicine, Loop of Henle, Animals, RNA, Messenger, Receptor, Tubuloglomerular feedback, Mice, Knockout, Multidisciplinary, Reverse Transcriptase Polymerase Chain Reaction, Hemodynamics, Receptors, Purinergic P1, Juxtaglomerular apparatus, Biological Sciences, Adenosine receptor, Juxtaglomerular Apparatus, Mice, Inbred C57BL, Arterioles, medicine.anatomical_structure, Endocrinology, Kidney Tubules, Vasoconstriction, Models, Animal, Macula densa, Female, Energy Metabolism, medicine.drug, Glomerular Filtration Rate
Abstract

Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR +/+ , A1AR +/− , and A1AR −/− mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR −/− mice (stop flow pressure response, −6.8 ± 0.55 mmHg and −0.4 ± 0.2 in A1AR +/+ and A1AR −/− mice; early proximal flow rate response, −3.4 ± 0.4 nl/min and +0.02 ± 0.3 nl/min in A1AR +/+ and A1AR −/− mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.

Published
2001

8. Translational control is required for the unfolded protein response and in vivo glucose homeostasis

Author

Chuan Liu, Donalyn Scheuner, Thom Saunders, Susan Bonner-Weir, Benbo Song, Randal J. Kaufman, Edward L. McEwen, Patrick J. Gillespie, and Ross Laybutt

Subjects
Transcriptional Activation, Protein Folding, Cell Survival, Gene Expression, Biology, Endoplasmic Reticulum, environment and public health, Islets of Langerhans, Mice, Eukaryotic translation, Translational regulation, Glucose homeostasis, Initiation factor, Animals, Homeostasis, EIF2AK3, RNA, Messenger, Phosphorylation, Molecular Biology, Endoplasmic Reticulum Chaperone BiP, Germ-Line Mutation, Heat-Shock Proteins, Endoplasmic reticulum, Homozygote, Gluconeogenesis, Cell Biology, Blood Proteins, Molecular biology, Activating Transcription Factors, Hypoglycemia, Mice, Mutant Strains, DNA-Binding Proteins, enzymes and coenzymes (carbohydrates), Glucose, Animals, Newborn, Mutagenesis, Protein Biosynthesis, Unfolded protein response, CCAAT-Enhancer-Binding Proteins, Carrier Proteins, Transcription Factor CHOP, Molecular Chaperones, Transcription Factors
Abstract

The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2alpha phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2alpha phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic beta cells. The results demonstrate that regulation of translation through eIF2alpha phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.

Published
2001

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