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Methods in Cell Biology 2016We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method...
We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture.
Topics: Animals; Calcium Signaling; Cell Culture Techniques; Cells, Cultured; Fluorescent Antibody Technique; Gene Knockdown Techniques; Hearing Loss, Sensorineural; Mice; MicroRNAs; Myosin Heavy Chains; Myosin Type V; Piebaldism; Pigmentation Disorders; Plasmids; Promoter Regions, Genetic; Purkinje Cells; Transfection
PubMed: 26794514
DOI: 10.1016/bs.mcb.2015.06.004 -
Frontiers in Veterinary Science 2015Although deafness can be acquired throughout an animal's life from a variety of causes, hereditary deafness, especially congenital hereditary deafness, is a significant... (Review)
Review
Although deafness can be acquired throughout an animal's life from a variety of causes, hereditary deafness, especially congenital hereditary deafness, is a significant problem in several species. Extensive reviews exist of the genetics of deafness in humans and mice, but not for deafness in domestic animals. Hereditary deafness in many species and breeds is associated with loci for white pigmentation, where the cochlear pathology is cochleo-saccular. In other cases, there is no pigmentation association and the cochlear pathology is neuroepithelial. Late onset hereditary deafness has recently been identified in dogs and may be present but not yet recognized in other species. Few genes responsible for deafness have been identified in animals, but progress has been made for identifying genes responsible for the associated pigmentation phenotypes. Across species, the genes identified with deafness or white pigmentation patterns include MITF, PMEL, KIT, EDNRB, CDH23, TYR, and TRPM1 in dog, cat, horse, cow, pig, sheep, ferret, mink, camelid, and rabbit. Multiple causative genes are present in some species. Significant work remains in many cases to identify specific chromosomal deafness genes so that DNA testing can be used to identify carriers of the mutated genes and thereby reduce deafness prevalence.
PubMed: 26664958
DOI: 10.3389/fvets.2015.00029 -
Genetics Dec 2015In population genomics studies, accounting for the neutral covariance structure across population allele frequencies is critical to improve the robustness of genome-wide...
In population genomics studies, accounting for the neutral covariance structure across population allele frequencies is critical to improve the robustness of genome-wide scan approaches. Elaborating on the BayEnv model, this study investigates several modeling extensions (i) to improve the estimation accuracy of the population covariance matrix and all the related measures, (ii) to identify significantly overly differentiated SNPs based on a calibration procedure of the XtX statistics, and (iii) to consider alternative covariate models for analyses of association with population-specific covariables. In particular, the auxiliary variable model allows one to deal with multiple testing issues and, providing the relative marker positions are available, to capture some linkage disequilibrium information. A comprehensive simulation study was carried out to evaluate the performances of these different models. Also, when compared in terms of power, robustness, and computational efficiency to five other state-of-the-art genome-scan methods (BayEnv2, BayScEnv, BayScan, flk, and lfmm), the proposed approaches proved highly effective. For illustration purposes, genotyping data on 18 French cattle breeds were analyzed, leading to the identification of 13 strong signatures of selection. Among these, four (surrounding the KITLG, KIT, EDN3, and ALB genes) contained SNPs strongly associated with the piebald coloration pattern while a fifth (surrounding PLAG1) could be associated to morphological differences across the populations. Finally, analysis of Pool-Seq data from 12 populations of Littorina saxatilis living in two different ecotypes illustrates how the proposed framework might help in addressing relevant ecological issues in nonmodel species. Overall, the proposed methods define a robust Bayesian framework to characterize adaptive genetic differentiation across populations. The BayPass program implementing the different models is available at http://www1.montpellier.inra.fr/CBGP/software/baypass/.
Topics: Adaptation, Biological; Animals; Cattle; Computer Simulation; Female; Genetic Variation; Genetics, Population; Genome; Genome, Human; Genotype; Humans; Male; Models, Genetic; Selection, Genetic
PubMed: 26482796
DOI: 10.1534/genetics.115.181453 -
Therapeutics and Clinical Risk... 2015Piebaldism is a rare autosomal dominant genodermatosis, manifesting as congenital and stable depigmentation of the skin and white forelock. It has been found to be...
Piebaldism is a rare autosomal dominant genodermatosis, manifesting as congenital and stable depigmentation of the skin and white forelock. It has been found to be associated with mutations in the KIT or SLUG genes. We report a Chinese piebaldism family including a 28-year-old woman and her 3-year-old son with characteristics of white patches and forelock associated with numerous brown macules and patches. Genomic DNA samples of the proband and her son were extracted from their peripheral blood. One hundred unrelated healthy individuals were used as controls. All coding regions of KIT, SLUG, and NF1 genes were amplified by polymerase chain reaction using exon flanking intronic primers and Sanger sequencings were performed. DNA sequencing revealed heterozygous missense c.2431T>G mutation in exon 17 of the KIT gene in the proband and the affected son. No potentially pathogenic variant was identified in SLUG or NF1 genes. The nucleotide substitution was not found in 100 unrelated control individuals. This study reveals a novel KIT mutation in piebaldism, and it further supports that café-au-lait macules and intertriginous freckling of piebaldism are parts of pigmented anomaly in piebaldism, which does not necessarily represent coexistence of neurofibromatosis type 1 (NF1).
PubMed: 25960657
DOI: 10.2147/TCRM.S75544 -
Frontiers in Bioscience (Elite Edition) Jan 2015The mechanisms of asymmetric organ development have been under intensive investigation for years, yet the proposed mechanisms remain controversial (1-3). The female... (Review)
Review
The mechanisms of asymmetric organ development have been under intensive investigation for years, yet the proposed mechanisms remain controversial (1-3). The female Bruchus quadrimaculatus beetle insect develops two black-colored spots bilaterally located on each upper elytra wing by an unknown mechanism. Fifty percent of the P (for piebald, two colors) gene homozygous mutant insects, described in 1925, had a normal left elytrum (with two black spots) and an abnormal right elytrum (with two red spots) and the balance supported the converse lateralized pigment arrangement (4). Rather than supporting the conventional morphogen model for the wings pigmentation development, their biological origin is explained here with the somatic strand-specific epigenetic imprinting and selective sister chromatid segregation (SSIS) mechanism (5). We propose that the P gene product performs the selective sister chromatid segregation function to produce symmetric cell division of a specific cell during embryogenesis to result in the bilateral symmetric development of elytra black color spots and that the altered chromatid segregation pattern of the mutant causes asymmetric cell division to confer the piebald phenotype.
Topics: Animals; Chromatids; Chromosome Segregation; Coleoptera; Epigenesis, Genetic; Models, Genetic; Morphogenesis; Phenotype; Pigmentation; Schizosaccharomyces; Wings, Animal
PubMed: 25553380
DOI: 10.2741/E734 -
The Journal of Veterinary Medical... Feb 2015The classic piebald mutation in the endothelin receptor type B (Ednrb) gene was found on rolling Nagoya genetic background (PROD-s/s) mice with white coat spotting. To...
The classic piebald mutation in the endothelin receptor type B (Ednrb) gene was found on rolling Nagoya genetic background (PROD-s/s) mice with white coat spotting. To examine whether genetic background influenced the phenotype in the piebald mutant mice, we generated a congenic strain (B6.PROD-s/s), produced by repeated backcrosses to the C57BL/6J (B6) strain. Although B6.PROD-s/s mice showed white coat spotting, 7% of B6.PROD-s/s mice died between 2 and 5 weeks after birth due to megacolon. The PROD-s/s, s/s and Japanese fancy mouse 1 (JF1) strains, which also have piebald mutations on different genetic backgrounds with B6, showed only pigmentation defects without megacolon. In expression analyses, rectums of B6.PROD-s/s with megacolon mice showed ~5% of the level of Ednrb gene expression versus B6 mice. In histological analyses, aganglionosis was detected in the rectum of megacolon animals. The aganglionic rectum was thought to lead to severe constipation and intestinal blockage, resulting in megacolon. We also observed an abnormal intestinal flora, including a marked increase in Bacteroidaceae and Erysipelotrichaceae and a marked decrease in Lactobacillus and Clostridiales, likely inducing endotoxin production and a failure of the mucosal barrier system, leading ultimately to death. These results indicate that the genetic background plays a key role in the development of enteric ganglion neurons, controlled by the Ednrb gene, and that B6 has modifier gene (s) regarding aganglionosis.
Topics: Animals; DNA; Female; Gene Expression Regulation; Genotype; Male; Megacolon; Mice; Mice, Inbred C57BL; Microsatellite Repeats; Mutation; Piebaldism; RNA, Messenger; Receptor, Endothelin B
PubMed: 25328005
DOI: 10.1292/jvms.14-0408 -
BMJ Case Reports Oct 2014Griscelli syndrome type 2 is characterised by partial albinism and primary immunodeficiency. We present a case of a 3-year-old girl diagnosed with cerebellar involvement...
Griscelli syndrome type 2 is characterised by partial albinism and primary immunodeficiency. We present a case of a 3-year-old girl diagnosed with cerebellar involvement of Griscelli syndrome type 2. Neurological complications may accompany Griscelli syndrome, however, to the best of my knowledge there are only a few case reports of cerebellar involvement of Griscelli syndrome type 2 in the literature.
Topics: Cerebellar Diseases; Child, Preschool; Fatal Outcome; Female; Humans; Immunologic Deficiency Syndromes; Lymphohistiocytosis, Hemophagocytic; Piebaldism; Primary Immunodeficiency Diseases
PubMed: 25315806
DOI: 10.1136/bcr-2014-206703 -
Microsurgery Jan 2015Vascular endothelial growth factor (VEGF) induces angiogenesis and osteogenesis in bone allotransplants. We aim to determine whether bone remodeling in VEGF-treated bone...
BACKGROUND
Vascular endothelial growth factor (VEGF) induces angiogenesis and osteogenesis in bone allotransplants. We aim to determine whether bone remodeling in VEGF-treated bone allotransplants results from repopulation with circulation-derived autogenous cells or survival of allogenic transplant-derived cells.
METHODS
Vascularized femoral bone transplants were transplanted from female Dark Agouti rats (DA;RT1(a) ) to male Piebald Viral Glaxo (PVG;RT1(c) ). Arteriovenous bundle implantation and short-term immunosuppression were used to maintain cellular viability. VEGF was encapsulated in biodegradable microspheres and delivered intramedullary in the experimental group (n = 22). In the control group (n = 22), no VEGF was delivered. Rats were sacrificed at 4 or 18 weeks. Laser capture microdissection of bone remodeling areas was performed at the inner and outer cortex. Sex-mismatched genes were quantified with reverse transcription-polymerase chain reaction to determine the amount of male cells to total cells, defined as the relative expression ratio (rER).
RESULTS
At 4 weeks, rER was significantly higher at the inner cortex in VEGF-treated transplants as compared to untreated transplants (0.622 ± 0.225 vs. 0.362 ± 0.081, P = 0.043). At 4 weeks, the outer cortex in the control group had a significantly higher rER (P = 0.038), whereas in the VEGF group, the inner cortex had a higher rER (P = 0.015). Over time, in the outer cortex the rER significantly increased to 0.634 ± 0.106 at 18 weeks in VEGF-treated rats (P = 0.049). At 18 weeks, the rER was >0.5 at all cortical areas in both groups.
CONCLUSIONS
These in vivo findings suggest a chemotactic effect of intramedullary applied VEGF on recipient-derived bone and could imply that more rapid angiogenesis of vascularized allotransplants can be established with microencapsulated VEGF.
Topics: Animals; Bone Transplantation; Bone and Bones; Capsules; Female; Male; Microsurgery; Neovascularization, Physiologic; Rats; Rats, Inbred Strains; Transplantation Chimera; Vascular Endothelial Growth Factor A
PubMed: 25073635
DOI: 10.1002/micr.22300 -
Dermatology Online Journal Jul 2014Griscelli Syndrome (GS) is a rare autosomal recessive disorder characterized by pigmentary dilution of the hair and skin (partial albinism). Three different types (1-3)...
Griscelli Syndrome (GS) is a rare autosomal recessive disorder characterized by pigmentary dilution of the hair and skin (partial albinism). Three different types (1-3) caused by mutation in three different genes have been described. Patients with GS type 1 have primary central nervous system dysfunction; type 2 patients commonly develop hemophagocytic lymphohistiocytosis and type 3 patients present with partial albinism only. Two siblings discussed here had silvery hair, eyebrows and eyelashes since birth with no features suggestive of immunodeficiency or neurological impairment, making it an even rarer presentation of Griscelli Syndrome, type 3. Diagnosis was confirmed on light microscopy (LM) of hair shafts. Both GS1 and GS2 have been described earlier. However, extensive search of the literature failed to reveal a similar presentation from Indian origin. This is the first ever report of GS-3 in non-identical siblings from India.
Topics: Child; Child, Preschool; Diagnosis, Differential; Female; Humans; Male; Piebaldism; Pigmentation Disorders; Rare Diseases; Siblings; Skin
PubMed: 25046460
DOI: No ID Found -
Annals of Dermatology Apr 2014
PubMed: 24882989
DOI: 10.5021/ad.2014.26.2.264