Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

Kirov, Julia V.; Adkisson, Michael; Nava, Alessia; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, Kevin C K; Jong, Pieter J. de; West, David B.

doi:10.1371/journal.pone.0134155

Cited by 3 publications

(1 citation statement)

References 47 publications

(47 reference statements)

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“…We confirmed the structure and correct integration of the lacZ reporter cassette at the Scx locus through Sanger sequencing of genomic PCR products. However, no specific beta-galactosidase reporter activity was detected in developing tendon tissues in the heterozygous and homozygous mutant mouse embryos, likely due to reporter gene silencing described previously in multiple other KOMP mouse lines (Kirov et al, 2015). To confirm that Scx function is lost in the Scx −/− embryos, we carried out in situ hybridization assay using a probe specific for the Scx coding sequence and found that Scx mRNA expression was completely absent in the Scx −/− embryos (Figures 3B,C).…”

Section: Characterization Of a New Scx Mutant Mouse Resourcementioning

confidence: 73%

The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation

Liu

Lan

et al. 2021

Front. Cell Dev. Biol.

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Proper development of tendons is crucial for the integration and function of the musculoskeletal system. Currently little is known about the molecular mechanisms controlling tendon development and tendon cell differentiation. The transcription factor Scleraxis (Scx) is expressed throughout tendon development and plays essential roles in both embryonic tendon development and adult tendon healing, but few direct target genes of Scx in tendon development have been reported and genome-wide identification of Scx direct target genes in vivo has been lacking. In this study, we have generated a ScxFlag knockin mouse strain, which produces fully functional endogenous Scx proteins containing a 2xFLAG epitope tag at the carboxy terminus. We mapped the genome-wide Scx binding sites in the developing limb tendon tissues, identifying 12,097 high quality Scx regulatory cis-elements in-around 7,520 genes. Comparative analysis with previously reported embryonic tendon cell RNA-seq data identified 490 candidate Scx direct target genes in early tendon development. Furthermore, we characterized a new Scx gene-knockout mouse line and performed whole transcriptome RNA sequencing analysis of E15.5 forelimb tendon cells from Scx–/– embryos and control littermates, identifying 68 genes whose expression in the developing tendon tissues significantly depended on Scx function. Combined analysis of the ChIP-seq and RNA-seq data yielded 32 direct target genes that required Scx for activation and an additional 17 target genes whose expression was suppressed by Scx during early tendon development. We further analyzed and validated Scx-dependent tendon-specific expression patterns of a subset of the target genes, including Fmod, Kera, Htra3, Ssc5d, Tnmd, and Zfp185, by in situ hybridization and real-time quantitative polymerase chain reaction assays. These results provide novel insights into the molecular mechanisms mediating Scx function in tendon development and homeostasis. The ChIP-seq and RNA-seq data provide a rich resource for aiding design of further studies of the mechanisms regulating tendon cell differentiation and tendon tissue regeneration. The ScxFlag mice provide a valuable new tool for unraveling the molecular mechanisms involving Scx in the protein interaction and gene-regulatory networks underlying many developmental and disease processes.

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Section: Characterization Of a New Scx Mutant Mouse Resourcementioning

confidence: 73%

The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation

Liu

Lan

et al. 2021

Front. Cell Dev. Biol.

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Insulin-Deficient Mouseβ-Cells Do Not Fully Mature but Can Be Remedied Through Insulin Replacement by Islet Transplantation

2017

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Insulin receptor (IR) insufficiency in β-cells leads to impaired insulin secretion and reduced β-cell hyperplasia in response to hyperglycemia. Selective IR deficiency in β-cells in later embryological development may lead to compensatory β-cell hyperplasia. Although these findings suggest insulin signaling on the β-cell is important for β-cell function, they are confounded by loss of signaling by the insulinlike growth factors through the IR. To determine whether insulin itself is necessary for β-cell development and maturation, we performed a characterization of pancreatic islets in mice with deletions of both nonallelic insulin genes (Ins1-/-Ins2-/-). We immunostained neonatal Ins1-/-Ins2-/- and Ins1+/+Ins2+/+ pancreata and performed quantitative polymerase chain reaction on isolated neonatal islets. Insulin-deficient islets had reduced expression of factors normally expressed in maturing β-cells, including muscoloaponeurotic fibrosarcoma oncogene homolog A, homeodomain transcription factor 6.1, and glucose transporter 2. Ins1-/-Ins2-/-β-cells expressed progenitor factors associated with stem cells or dedifferentiated β-cells, including v-myc avian myolocytomatosis viral oncogene lung carcinoma derived and homeobox protein NANOG. We replaced insulin by injection or islet transplantation to keep mice alive into adulthood to determine whether insulin replacement was sufficient for the completed maturation of insulin-deficient β-cells. Short-term insulin glargine (Lantus®) injections partially rescued the β-cell phenotype, whereas long-term replacement of insulin by isogenic islet transplantation supported the formation of more mature β-cells. Our findings suggest that tightly regulated glycemia, insulin species, or other islet factors are necessary for β-cell maturation.

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Localization of Glucagon-Like Peptide-2 Receptor Expression in the Mouse

et al. 2019

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Glucagon-like peptide-2 (GLP-2), secreted from enteroendocrine cells, attenuates gut motility, enhances barrier function, and augments nutrient absorption, actions mediated by a single GLP-2 receptor (GLP-2R). Despite extensive analyses, the precise distribution and cellular localization of GLP-2R expression remains controversial, confounded by the lack of suitable GLP-2R antisera. Here, we reassessed murine Glp2r expression using regular and real-time quantitative PCR (qPCR), in situ hybridization (ISH), and a Glp2rLacZ reporter mouse. Glp2r mRNA expression was detected from the stomach to the rectum and most abundant in the jejunum. Glp2r transcripts were also detected in cerebral cortex, mesenteric lymph nodes, gallbladder, urinary bladder, and mesenteric fat. Surprisingly, Glp2r mRNA was found in testis by qPCR at levels similar to jejunum. However, the testis Glp2r transcripts, detected by different primer pairs and qPCR, lacked 5′ mRNA coding sequences, and only a minute proportion of them corresponded to full-length Glp2r mRNA. Within the gut, Glp2r-driven LacZ expression was localized to enteric neurons and lamina propria stromal cells, findings confirmed by ISH analysis of the endogenous Glp2r mRNA. Unexpectedly, vascular Glp2rLacZ expression was localized to mesenteric veins and not arteries. Moreover, mesenteric fat Glp2rLacZ expression was detected within blood vessels and not adipocytes. Reporter LacZ expression was not detected in all tissues expressing an endogenous Glp2r transcript, such as gallbladder, urinary bladder, and mesenteric lymph nodes. Collectively, these findings extend our understanding of the cellular domains of Glp2r expression and highlight limitations inherent in application of commonly used technologies to infer analysis of gene expression.

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Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

Cited by 3 publications

References 47 publications

The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation

The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation

Insulin-Deficient Mouseβ-Cells Do Not Fully Mature but Can Be Remedied Through Insulin Replacement by Islet Transplantation

Localization of Glucagon-Like Peptide-2 Receptor Expression in the Mouse

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