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1.
CBE Life Sci Educ ; 20(3): ar46, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34415196

RESUMEN

Course-based undergraduate research experiences (CUREs) engage students in authentic research experiences in a course format and can sometimes result in the publication of that research. However, little is known about student-author perceptions of CURE publications. In this study, we examined how students perceive they benefit from authoring a CURE publication and what they believe is required for authorship of a manuscript in a peer-reviewed journal. All 16 students who were enrolled in a molecular genetics CURE during their first year of college participated in semistructured interviews during their fourth year. At the time of the interviews, students had been authors of a CURE publication for a year and a half. Students reported that they benefited personally and professionally from the publication. Students had varying perceptions of what is required for authorship, but every student thought that writing the manuscript was needed, and only two mentioned needing to approve the final draft. Additionally, we identified incomplete conceptions that students had about CURE publications. This work establishes student-perceived benefits from CURE publications and highlights the need for authorship requirements to be explicitly addressed in CUREs.


Asunto(s)
Estudiantes , Universidades , Autoria , Humanos , Percepción
2.
MethodsX ; 8: 101419, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34430314

RESUMEN

Successful use of the CRISPR-Cas9 system for gene manipulation relies on identifying effective and efficient guide RNA sequences (gRNAs). When the goal is to create transgenic animal/rodent models by knocking-in desired sequences using homology-directed repair (HDR), selecting effective guides becomes even more critical to minimize developmental time and resources. Currently, validation experiments for gRNAs for generating rat models are carried out using immortalized rat cells. However, there are several limitations with using such cell lines, including ploidy of the genome, non-predictive transfection efficiency, and the ability to identify gene modifications efficiently within diverse cell populations. Since embryos are authentic representatives of live animals compared to cell lines, validating CRISPR guides for their nuclease activity in freshly isolated embryos will provide greater accuracy of in vivo gene editing efficiency. In contrast to microinjections, delivery by electroporation is a more accessible method that can be simple and does not require special skills and equipment. We demonstrate an accessible workflow to either delete or edit target genes in vivo in rats using the efficient editing of a human mutation in alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) ortholog using electroporation as a delivery method for CRISPR-Cas9 ribonucleoprotein complexes in rat embryos.•Upon identifying CRISPR targets at the desired genetic alteration site, we designed homologydriven repair (HDR) templates for effective and easy identification of gene editing by Restriction Fragment Length Polymorphism (RFLP).•Cultured rat embryos can be genotyped to assess CRISPR activity as seen by either presence of indels resulting from NHEJ or knock-in of repair template resulting from homology driven repair.•Heteroduplex mobility assay (HMA) and Restriction Fragment Length Polymorphism (RFLP) of PCR products can be performed reliably and reproducibly at a low-cost.

3.
Sci Rep ; 11(1): 11977, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34099738

RESUMEN

Although recent advances in the treatment of acute coronary heart disease have reduced mortality rates, few therapeutic strategies exist to mitigate the progressive loss of cardiac function that manifests as heart failure. Nuclear factor, erythroid 2 like 2 (Nfe2l2, Nrf2) is a transcriptional regulator that is known to confer transient myocardial cytoprotection following acute ischemic insult; however, its sustained activation paradoxically causes a reductive environment characterized by excessive antioxidant activity. We previously identified a subset of 16 microRNAs (miRNA) significantly diminished in Nrf2-ablated (Nrf2-/-) mouse hearts, leading to the hypothesis that increasing levels of Nrf2 activation augments miRNA induction and post-transcriptional dysregulation. Here, we report the identification of distinct miRNA signatures (i.e. "reductomiRs") associated with Nrf2 overexpression in a cardiac-specific and constitutively active Nrf2 transgenic (caNrf2-Tg) mice expressing low (TgL) and high (TgH) levels. We also found several Nrf2 dose-responsive miRNAs harboring proximal antioxidant response elements (AREs), implicating these "reductomiRs" as putative meditators of Nrf2-dependent post-transcriptional regulation. Analysis of mRNA-sequencing identified a complex network of miRNAs and effector mRNAs encoding known pathological hallmarks of cardiac stress-response. Altogether, these data support Nrf2 as a putative regulator of cardiac miRNA expression and provide novel candidates for future mechanistic investigation to understand the relationship between myocardial reductive stress and cardiac pathophysiology.


Asunto(s)
Biomarcadores/metabolismo , Corazón/fisiología , MicroARNs/metabolismo , Miocardio/metabolismo , Factor 2 Relacionado con NF-E2/genética , Animales , Antioxidantes , Secuencia de Bases , Citoprotección , Regulación de la Expresión Génica , Insuficiencia Cardíaca , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Transducción de Señal
4.
Zebrafish ; 18(4): 293-296, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34030492

RESUMEN

Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. It can arise in vascular or lymphatic tissues, involve any part of the body, and aggressively spread locally or metastasize. Angiosarcomas spontaneously develop in the tp53 deleted (tp53del/del) zebrafish mutant. However, established protocols for tumor dissection and transplantation of single cell suspensions of angiosarcoma tumors result in inferior implantation rates. To resolve these complications, we developed a new tumor grafting technique for engraftment of angiosarcoma and similar tumors in zebrafish, which maintains the tumor microenvironment and has superior rates of engraftment.

5.
Aging Cell ; 20(4): e13339, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33755309

RESUMEN

Mice with disruptions of growth hormone-releasing hormone (GHRH) or growth hormone receptor (GHR) exhibit similar phenotypes of prolonged lifespan and delayed age-related diseases. However, these two models respond differently to calorie restriction indicating that they might carry different and/or independent mechanisms for improved longevity and healthspan. In order to elucidate these mechanisms, we generated GHRH and GHR double-knockout mice (D-KO). In the present study, we focused specifically on the characteristics of female D-KO mice. The D-KO mice have reduced body weight and enhanced insulin sensitivity compared to wild-type (WT) controls. Growth retardation in D-KO mice is accompanied by decreased GH expression in pituitary, decreased circulating IGF-1, increased high-molecular-weight (HMW) adiponectin, and leptin hormones compared to WT controls. Generalized linear model-based regression analysis, which controls for body weight differences between D-KO and WT groups, shows that D-KO mice have decreased lean mass, bone mineral density, and bone mineral content, but increased adiposity. Indirect calorimetry markers including oxygen consumption, carbon dioxide production, and energy expenditure were significantly lower in D-KO mice relative to the controls. In comparison with WT mice, the D-KO mice displayed reduced respiratory exchange ratio (RER) values only during the light cycle, suggesting a circadian-related metabolic shift toward fat utilization. Interestingly, to date survival data suggest extended lifespan in D-KO female mice.

6.
Dis Model Mech ; 13(11)2020 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-32928875

RESUMEN

L-2-hydroxyglutarate (L-2HG) is an oncometabolite found elevated in renal tumors. However, this molecule might have physiological roles that extend beyond its association with cancer, as L-2HG levels are elevated in response to hypoxia and during Drosophila larval development. L-2HG is known to be metabolized by L-2HG dehydrogenase (L2HGDH), and loss of L2HGDH leads to elevated L-2HG levels. Despite L2HGDH being highly expressed in the kidney, its role in renal metabolism has not been explored. Here, we report our findings utilizing a novel CRISPR/Cas9 murine knockout model, with a specific focus on the role of L2HGDH in the kidney. Histologically, L2hgdh knockout kidneys have no demonstrable histologic abnormalities. However, GC-MS metabolomics demonstrates significantly reduced levels of the TCA cycle intermediate succinate in multiple tissues. Isotope labeling studies with [U-13C] glucose demonstrate that restoration of L2HGDH in renal cancer cells (which lowers L-2HG) leads to enhanced incorporation of label into TCA cycle intermediates. Subsequent biochemical studies demonstrate that L-2HG can inhibit the TCA cycle enzyme α-ketoglutarate dehydrogenase. Bioinformatic analysis of mRNA expression data from renal tumors demonstrates that L2HGDH is co-expressed with genes encoding TCA cycle enzymes as well as the gene encoding the transcription factor PGC-1α, which is known to regulate mitochondrial metabolism. Restoration of PGC-1α in renal tumor cells results in increased L2HGDH expression with a concomitant reduction in L-2HG levels. Collectively, our analyses provide new insight into the physiological role of L2HGDH as well as mechanisms that promote L-2HG accumulation in disease states.


Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Riñón/enzimología , Oxidorreductasas de Alcohol/genética , Animales , Encéfalo/enzimología , Encéfalo/patología , Sistemas CRISPR-Cas/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Ciclo del Ácido Cítrico , Fertilidad , Regulación Neoplásica de la Expresión Génica , Glutaratos/metabolismo , Heterocigoto , Riñón/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Análisis de Flujos Metabólicos , Metaboloma , Metabolómica , Ratones Noqueados , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ácido Succínico/metabolismo
7.
Artículo en Inglés | MEDLINE | ID: mdl-31160943

RESUMEN

As CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology becomes more mainstream in life science research, it becomes critical for undergraduate instructors to devise engaging ways to bring the technology into their classrooms. To help meet this challenge, the National Science Foundation sponsored a workshop for undergraduate instructors in June 2018 at The Ohio State University in conjunction with the annual Association of Biology Laboratory Educators meeting based on a workflow developed by the workshop's facilitators. Over the course of two and a half days, participants worked through a modular workflow for the use of CRISPR-Cas9 in a course-based (undergraduate) research experience (CURE) setting while discussing the barriers each of their institutions had to implementing such work, and how such barriers could be overcome. The result of the workshop was a team with newfound energy and confidence to implement CRISPR-Cas9 technology in their courses and the development of a community of undergraduate educators dedicated to supporting each other in the implementation of the workflow either in a CURE or modular format. In this article, we review the activities and discussions from the workshop that helped each participant devise their own tailored approaches of how best to bring this exciting new technology into their classes.

8.
Front Cardiovasc Med ; 6: 68, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31245386

RESUMEN

Although exercise derived activation of Nrf2 signaling augments myocardial antioxidant signaling, the molecular mechanisms underlying the benefits of moderate exercise training (MET) in the heart remain elusive. Here we hypothesized that exercise training stabilizes Nrf2-dependent antioxidant signaling, which then protects the myocardium from isoproterenol-induced damage. The present study assessed the effects of 6 weeks of MET on the Nrf2/antioxidant function, glutathione redox state, and injury in the myocardium of C57/BL6J mice that received isoproterenol (ISO; 50 mg/kg/day for 7 days). ISO administration significantly reduced the Nrf2 promoter activity (p < 0.05) and downregulated the expression of cardiac antioxidant genes (Gclc, Nqo1, Cat, Gsr, and Gst-µ) in the untrained (UNT) mice. Furthermore, increased oxidative stress with severe myocardial injury was evident in UNT+ISO when compared to UNT mice receiving PBS under basal condition. Of note, MET stabilized the Nrf2-promoter activity and upheld the expression of Nrf2-dependent antioxidant genes in animals receiving ISO, and attenuated the oxidative stress-induced myocardial damage. Echocardiography analysis revealed impaired diastolic ventricular function in UNT+ISO mice, but this was partially normalized in the MET animals. Interestingly, while there was a marginal reduction in ubiquitinated proteins in MET mice that received ISO, the pathological signs were attenuated along with near normal cardiac function in response to exercise training. Thus, moderate intensity exercise training conferred protection against ISO-induced myocardial injury by augmentation of Nrf2-antioxidant signaling and attenuation of isoproterenol-induced oxidative stress.

9.
MethodsX ; 6: 1-5, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30591915

RESUMEN

The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.

10.
Am J Physiol Renal Physiol ; 316(3): F414-F425, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30566001

RESUMEN

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.


Asunto(s)
Metabolismo Energético/genética , Glucosa/metabolismo , Proteínas Quinasas/genética , Receptores de Superficie Celular/genética , Proliferación Celular/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Células HEK293 , Humanos , Mutación , Proteínas Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo
11.
J Genet ; 97(5): 1315-1325, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30555080

RESUMEN

Nodal-related protein (ndr2) is amember of the transforming growth factor type ß superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.


Asunto(s)
Sistemas CRISPR-Cas , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Ribonucleoproteínas/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Holoprosencefalia/genética , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Fenotipo , Dominios Proteicos , Ribonucleoproteínas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química
12.
NPJ Breast Cancer ; 4: 29, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30182054

RESUMEN

The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR-Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.

13.
Dis Model Mech ; 9(10): 1169-1179, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27483347

RESUMEN

Osteocalcin, also known as bone γ-carboxyglutamate protein (Bglap), is expressed by osteoblasts and is commonly used as a clinical marker of bone turnover. A mouse model of osteocalcin deficiency has implicated osteocalcin as a mediator of changes to the skeleton, endocrine system, reproductive organs and central nervous system. However, differences between mouse and human osteocalcin at both the genome and protein levels have challenged the validity of extrapolating findings from the osteocalcin-deficient mouse model to human disease. The rat osteocalcin (Bglap) gene locus shares greater synteny with that of humans. To further examine the role of osteocalcin in disease, we created a rat model with complete loss of osteocalcin using the CRISPR/Cas9 system. Rat osteocalcin was modified by injection of CRISPR/Cas9 mRNA into the pronuclei of fertilized single cell Sprague-Dawley embryos, and animals were bred to homozygosity and compound heterozygosity for the mutant alleles. Dual-energy X-ray absorptiometry (DXA), glucose tolerance testing (GTT), insulin tolerance testing (ITT), microcomputed tomography (µCT), and a three-point break biomechanical assay were performed on the excised femurs at 5 months of age. Complete loss of osteocalcin resulted in bones with significantly increased trabecular thickness, density and volume. Cortical bone volume and density were not increased in null animals. The bones had improved functional quality as evidenced by an increase in failure load during the biomechanical stress assay. Differences in glucose homeostasis were observed between groups, but there were no differences in body weight or composition. This rat model of complete loss of osteocalcin provides a platform for further understanding the role of osteocalcin in disease, and it is a novel model of increased bone formation with potential utility in osteoporosis and osteoarthritis research.


Asunto(s)
Sistemas CRISPR-Cas/genética , Hueso Esponjoso/fisiología , Osteocalcina/deficiencia , Absorciometría de Fotón , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Fenómenos Biomecánicos , Composición Corporal , Hueso Esponjoso/diagnóstico por imagen , Fémur/diagnóstico por imagen , Fémur/fisiología , Efecto Fundador , Técnicas Genéticas , Prueba de Tolerancia a la Glucosa , Mutación INDEL/genética , Insulina/metabolismo , Masculino , Modelos Animales , Osteocalcina/química , Osteocalcina/metabolismo , Ratas , Especificidad de la Especie , Microtomografía por Rayos X
14.
PLoS Genet ; 12(7): e1006220, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27472056

RESUMEN

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.


Asunto(s)
Tipificación del Cuerpo/genética , Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animales , Movimiento Celular/genética , Chlamydomonas/genética , Cilios/patología , Proteínas del Citoesqueleto , Citoesqueleto/genética , Modelos Animales de Enfermedad , Extremidades/crecimiento & desarrollo , Extremidades/patología , Predisposición Genética a la Enfermedad , Humanos , Síndrome de Kartagener/patología , Ratones , Microtúbulos/genética , Mutación , Tubo Neural/crecimiento & desarrollo , Tubo Neural/patología , Transducción de Señal/genética
15.
PLoS One ; 11(5): e0155812, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27224051

RESUMEN

Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (Tyr) cause oculocutaneous albinism (OCA1) in humans. Alleles of the Tyr gene have been useful in studying pigment biology and coat color formation. Over 100 different Tyr alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted a priori. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an in vivo visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of Tyr, dhoosara (Sanskrit for gray) and chandana (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of Tyr gene.


Asunto(s)
Alelos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Color del Cabello/genética , Monofenol Monooxigenasa/genética , Eliminación de Secuencia , Animales , Femenino , Masculino , Ratones
16.
J Biol Chem ; 281(12): 7825-33, 2006 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-16434401

RESUMEN

Sialic acids are sometimes 9-O-acetylated in a developmentally regulated and cell-type-specific manner. Cells naturally expressing the disialoganglioside GD3 often O-acetylate the terminal sialic acid residue, giving 9-O-acetyl-GD3 (9AcGD3), a marker of neural differentiation and malignant transformation. We also reported that Chinese hamster ovary cells transfected with GD3 synthase can spontaneously O-acetylate some of the newly synthesized GD3. It is unclear whether such phenomena result from induction of the 9-O-acetylation machinery and whether induction is caused by the GD3 synthase protein or by the GD3 molecule itself. We now show that exogenously added GD3 rapidly incorporates into the plasma membrane of Chinese hamster ovary cells, and 9AcGD3 is detected after approximately 6 h. The incorporated GD3 and newly synthesized 9AcGD3 have a half-life of approximately 24 h. This phenomenon is also seen in other cell types, such as human diploid fibroblasts. Inhibitors of gene transcription, protein translation, or endoplasmic reticulum-to-Golgi transport each prevent induction of 9-O-acetylation, without affecting GD3 incorporation. Inhibition of the initial clathrin-independent internalization of incorporated GD3 also blocks induction of 9-O-acetylation. Thus, new synthesis of one or more components of the 9-O-acetylation machinery is induced by incorporation and internalization of GD3. Prepriming with structurally related gangliosides fails to accelerate the onset of 9-O-acetylation of subsequently added GD3, indicating a requirement for specific recognition of GD3. To our knowledge, this is the first example wherein a newly expressed or exogenously introduced ganglioside induces de novo synthesis of an enzymatic machinery to modify itself, and the first evidence for a mechanism of induction of sialic acid O-acetylation.


Asunto(s)
Gangliósidos/química , Acetilación , Animales , Células CHO , Diferenciación Celular , Línea Celular , Membrana Celular/metabolismo , Transformación Celular Neoplásica , Clatrina/metabolismo , Cricetinae , Medios de Cultivo/metabolismo , Medio de Cultivo Libre de Suero/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Endocitosis , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Citometría de Flujo , Aparato de Golgi/metabolismo , Humanos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Neuronas/metabolismo , Unión Proteica , Ácidos Siálicos/química , Factores de Tiempo , Transcripción Genética , Transfección
17.
Mech Dev ; 122(10): 1073-86, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16129585

RESUMEN

Roundabout (Robo) receptors and their secreted ligand Slits have been shown to function in a number of developmental events both inside and outside of the nervous system. We previously cloned zebrafish robo orthologs to gain a better understanding of Robo function in vertebrates. Further characterization of one of these orthologs, robo3, has unveiled the presence of two distinct isoforms, robo3 variant 1 (robo3var1) and robo3 variant 2 (robo3var2). These two isoforms differ only in their 5'-ends with robo3var1, but not robo3var2, containing a canonical signal sequence. Despite this difference, both forms accumulate on the cell surface. Both isoforms are contributed maternally and exhibit unique and dynamic gene expression patterns during development. Functional analysis of robo3 isoforms using an antisense gene knockdown strategy suggests that Robo3var1 functions in motor axon pathfinding, whereas Robo3var2 appears to function in dorsoventral cell fate specification. This study reveals a novel function for Robo receptors in specifying ventral cell fates during vertebrate development.


Asunto(s)
Proteínas de Drosophila/fisiología , Sistema Nervioso/embriología , Receptores Inmunológicos/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Mutación , Sistema Nervioso/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores Inmunológicos/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética
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