scholarly journals Rod- and cone-driven responses in mice expressing human L-cone pigment

2015 ◽  
Vol 114 (4) ◽  
pp. 2230-2241 ◽  
Author(s):  
Tina I. Tsai ◽  
Jenny Atorf ◽  
Maureen Neitz ◽  
Jay Neitz ◽  
Jan Kremers
Keyword(s):  
Absorption Spectra ◽  
Genetic Manipulation ◽  
Temporal Frequency ◽  
Silent Substitution ◽  
Retinal Processing ◽  
Cone Pigment ◽  
Retinal Adaptation ◽  
Compensation Approach ◽  
Phase Behaviors

The mouse is commonly used for studying retinal processing, primarily because it is amenable to genetic manipulation. To accurately study photoreceptor driven signals in the healthy and diseased retina, it is of great importance to isolate the responses of single photoreceptor types. This is not easily achieved in mice because of the strong overlap of rod and M-cone absorption spectra (i.e., maxima at 498 and 508 nm, respectively). With a newly developed mouse model ( Opn1lwLIAIS) expressing a variant of the human L-cone pigment (561 nm) instead of the mouse M-opsin, the absorption spectra are substantially separated, allowing retinal physiology to be studied using silent substitution stimuli. Unlike conventional chromatic isolation methods, this spectral compensation approach can isolate single photoreceptor subtypes without changing the retinal adaptation. We measured flicker electroretinograms in these mutants under ketamine-xylazine sedation with double silent substitution (silent S-cone and either rod or M/L-cones) and obtained robust responses for both rods and (L-)cones. Small signals were yielded in wild-type mice, whereas heterozygotes exhibited responses that were generally intermediate to both. Fundamental response amplitudes and phase behaviors (as a function of temporal frequency) in all genotypes were largely similar. Surprisingly, isolated (L-)cone and rod response properties in the mutant strain were alike. Thus the LIAIS mouse warrants a more comprehensive in vivo assessment of photoreceptor subtype-specific physiology, because it overcomes the hindrance of overlapping spectral sensitivities present in the normal mouse.

Improvement of effectiveness in maize breeding

2006 ◽  
Vol 54 (3) ◽  
pp. 351-358 ◽  
Author(s):  
P. Pepó
Keyword(s):  
Recurrent Selection ◽  
Genetic Manipulation ◽  
Field Testing ◽  
Tissue Cultures ◽  
Maize Breeding ◽  
Breeding Programmes ◽  
Speed Up ◽  
Selection For

Plant regeneration via tissue culture is becoming increasingly more common in monocots such as maize (Zea mays L.). Pollen (gametophytic) selection for resistance to aflatoxin in maize can greatly facilitate recurrent selection and the screening of germplasm for resistance at much less cost and in a shorter time than field testing. In vivo and in vitro techniques have been integrated in maize breeding programmes to obtain desirable agronomic attributes, enhance the genes responsible for them and speed up the breeding process. The efficiency of anther and tissue cultures in maize and wheat has reached the stage where they can be used in breeding programmes to some extent and many new cultivars produced by genetic manipulation have now reached the market.

Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting

2005 ◽  
Vol 289 (1) ◽  
pp. E53-E61 ◽  
Author(s):  
Shawn C. Burgess ◽  
F. Mark H. Jeffrey ◽  
Charles Storey ◽  
Angela Milde ◽  
Natasha Hausler ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Glucose Production ◽  
Tca Cycle ◽  
Inbred Strains ◽  
Mouse Strains ◽  
Background Strain ◽  
Metabolic Fluxes ◽  
Inbred Strains Of Mice ◽  
Total Glucose

Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phospho enolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was ∼30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism.

scholarly journals Gene Transfer to the Desiccation-Tolerant CyanobacteriumChroococcidiopsis

2001 ◽  
Vol 183 (7) ◽  
pp. 2298-2305 ◽  
Author(s):  
Daniela Billi ◽  
E. Imre Friedmann ◽  
Richard F. Helm ◽  
Malcolm Potts
Keyword(s):  
High Efficiency ◽  
Genetic Manipulation ◽  
Recipient Cell ◽  
Conjugative Transfer ◽  
Lower Efficiency ◽  
Genetic Studies ◽  
Variable Regions ◽  
Multiple Cycles ◽  
Experimental Strain

ABSTRACT The coccoid cyanobacterium Chroococcidiopsisdominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation ofChroococcidiopsis. Plasmids pDUCA7 and pRL489, based on the pDU1 replicon of Nostoc sp. strain PCC 7524, were transferred to different isolates of Chroococcidiopsisvia conjugation and electroporation. This report provides the first evidence that pDU1 replicons can be maintained in cyanobacteria other than Nostoc and Anabaena. Following conjugation, both plasmids replicated inChroococcidiopsis sp. strains 029, 057, and 123 but not in strains 171 and 584. Both plasmids were electroporated into strains 029 and 123 but not into strains 057, 171, and 584. Expression of P psbA-luxAB on pRL489 was visualized through in vivo luminescence. Efficiencies of conjugative transfer for pDUCA7 and pRL489 into Chroococcidiopsissp. strain 029 were approximately 10−2 and 10−4 transconjugants per recipient cell, respectively. Conjugative transfer occurred with a lower efficiency into strains 057 and 123. Electrotransformation efficiencies of about 10−4electrotransformants per recipient cell were achieved with strains 029 and 123, using either pDUCA7 or pRL489. Extracellular deoxyribonucleases were associated with each of the five strains. Phylogenetic analysis, based upon the V6 to V8 variable regions of 16S rRNA, suggests that desert strains 057, 123, 171, and 029 are distinct from the type species strain Chroococcidiopsis thermalis PCC 7203. The high efficiency of conjugative transfer of Chroococcidiopsis sp. strain 029, from the Negev Desert, Israel, makes this a suitable experimental strain for genetic studies on desiccation tolerance.

Effect of ultraviolet adaptation on the ultraviolet absorption spectra of human skin in vivo

2008 ◽  
Vol 24 (2) ◽  
pp. 76-82 ◽  
Author(s):  
Merve Meinhardt ◽  
Ronald Krebs ◽  
Angelika Anders ◽  
Ulrike Heinrich ◽  
Hagen Tronnier
Keyword(s):  
Absorption Spectra ◽  
Human Skin ◽  
Ultraviolet Absorption

Dynamics of neurons in the cat lateral geniculate nucleus: in vivo electrophysiology and computational modeling

1995 ◽  
Vol 74 (3) ◽  
pp. 1222-1243 ◽  
Author(s):  
P. Mukherjee ◽  
E. Kaplan
Keyword(s):  
Computational Modeling ◽  
Spike Train ◽  
Lateral Geniculate Nucleus ◽  
Transfer Functions ◽  
Temporal Frequency ◽  
Relay Cell ◽  
Lateral Geniculate ◽  
Low Pass ◽  
Band Pass

1. We investigated the time domain transformation that thalamocortical relay cells of the cat lateral geniculate nucleus (LGN) perform on their retinal input, and used computational modeling to explore the biophysical properties that determine the dynamics of the LGN relay cells in vivo. 2. We recorded simultaneously the input (S potentials) and output (action potentials) of 50 cat LGN relay cells stimulated by drifting sinusoidal gratings of varying temporal frequency. The temporal modulation transfer functions (TMTFs) of the neurons were derived from these data. The burstiness of the LGN spike trains was also assessed using objective criteria. 3. We found that the form of the TMTF was quite variable among cells, ranging from low-pass to strongly band-pass. The optimal temporal frequency of band-pass neurons was between 2 and 8 Hz. In addition, the TMTF of some cells was nonstationary: their temporal tuning changed with time. 4. The temporal tuning of a cell was directly related to the degree of burstiness of its spike train. Tonically firing relay cells had low-pass TMTFs, whereas the most bursty neurons exhibited the most sharply band-pass transfer functions. This was also true for single cells that altered their temporal tuning: a shift to more band-pass tuning was associated with increased burstiness of the spike train, and vice versa. 5. We constructed a computer simulation of the LGN relay cell. The model was a simplified five-channel version of the thalamocortical neuron model of McCormick and Huguenard. It incorporated the quantitative kinetics of the Ca2+ T channel, as well as the Hodgkin-Huxley Na+ and K+ channels, as the only active membrane currents. To simulate the in vivo dynamics of the relay cell, the input to the model consisted of trains of synaptic potentials, recorded as S potentials in our physiological experiments. 6. When the resting membrane potential of the model neuron was relatively depolarized, the model's TMTF was low-pass, with no bursting evident in the simulated spike train. At hyperpolarized resting membrane potentials, however, the modeled TMTF was band-pass, with frequent burst discharges. Thus the biophysical model reproduced not only the range of dynamics seen in real LGN relay cells, but also the dependence of the overall dynamics on the burstiness of the spike train. However, neither of these phenomena could be simulated without the T channel. Thus the simulations demonstrated that the T-type Ca2+ channel was necessary and sufficient to explain the LGN dynamics observed in physiological experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The Ferret as a Model System for Neocortex Development and Evolution

2021 ◽  
Vol 9 ◽  
Author(s):  
Carlotta Gilardi ◽  
Nereo Kalebic
Keyword(s):  
Animal Model ◽  
Cognitive Abilities ◽  
Genetic Manipulation ◽  
System Development ◽  
Model Organisms ◽  
Cortical Folding ◽  
Technological Advances ◽  
Stem And Progenitor Cells ◽  
Visual System Development

The neocortex is the largest part of the cerebral cortex and a key structure involved in human behavior and cognition. Comparison of neocortex development across mammals reveals that the proliferative capacity of neural stem and progenitor cells and the length of the neurogenic period are essential for regulating neocortex size and complexity, which in turn are thought to be instrumental for the increased cognitive abilities in humans. The domesticated ferret, Mustela putorius furo, is an important animal model in neurodevelopment for its complex postnatal cortical folding, its long period of forebrain development and its accessibility to genetic manipulation in vivo. Here, we discuss the molecular, cellular, and histological features that make this small gyrencephalic carnivore a suitable animal model to study the physiological and pathological mechanisms for the development of an expanded neocortex. We particularly focus on the mechanisms of neural stem cell proliferation, neuronal differentiation, cortical folding, visual system development, and neurodevelopmental pathologies. We further discuss the technological advances that have enabled the genetic manipulation of the ferret in vivo. Finally, we compare the features of neocortex development in the ferret with those of other model organisms.

scholarly journals Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1526
Author(s):  
Joanna E. Kowalczyk ◽  
Shreya Saha ◽  
Miia R. Mäkelä
Keyword(s):  
Genome Editing ◽  
Plant Biomass ◽  
Genetic Manipulation ◽  
White Rot ◽  
White Rot Fungus ◽  
Detailed Knowledge ◽  
Wild Type ◽  
Low Frequencies ◽  
Dichomitus Squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

scholarly journals Zebrafish Cancer Predisposition Models

2021 ◽  
Vol 9 ◽  
Author(s):  
Kim Kobar ◽  
Keon Collett ◽  
Sergey V. Prykhozhij ◽  
Jason N. Berman
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Bone Marrow Failure ◽  
Cancer Predisposition ◽  
Potential Candidate ◽  
Cancer Evolution ◽  
High Fecundity ◽  
Cancer Models ◽  
The Impact

Cancer predisposition syndromes are rare, typically monogenic disorders that result from germline mutations that increase the likelihood of developing cancer. Although these disorders are individually rare, resulting cancers collectively represent 5–10% of all malignancies. In addition to a greater incidence of cancer, affected individuals have an earlier tumor onset and are frequently subjected to long-term multi-modal cancer screening protocols for earlier detection and initiation of treatment. In vivo models are needed to better understand tumor-driving mechanisms, tailor patient screening approaches and develop targeted therapies to improve patient care and disease prognosis. The zebrafish (Danio rerio) has emerged as a robust model for cancer research due to its high fecundity, time- and cost-efficient genetic manipulation and real-time high-resolution imaging. Tumors developing in zebrafish cancer models are histologically and molecularly similar to their human counterparts, confirming the validity of these models. The zebrafish platform supports both large-scale random mutagenesis screens to identify potential candidate/modifier genes and recently optimized genome editing strategies. These techniques have greatly increased our ability to investigate the impact of certain mutations and how these lesions impact tumorigenesis and disease phenotype. These unique characteristics position the zebrafish as a powerful in vivo tool to model cancer predisposition syndromes and as such, several have already been created, including those recapitulating Li-Fraumeni syndrome, familial adenomatous polyposis, RASopathies, inherited bone marrow failure syndromes, and several other pathogenic mutations in cancer predisposition genes. In addition, the zebrafish platform supports medium- to high-throughput preclinical drug screening to identify compounds that may represent novel treatment paradigms or even prevent cancer evolution. This review will highlight and synthesize the findings from zebrafish cancer predisposition models created to date. We will discuss emerging trends in how these zebrafish cancer models can improve our understanding of the genetic mechanisms driving cancer predisposition and their potential to discover therapeutic and/or preventative compounds that change the natural history of disease for these vulnerable children, youth and adults.

scholarly journals Induction and Maturation of Hepatocyte-Like Cells In Vitro: Focus on Technological Advances and Challenges

2021 ◽  
Vol 9 ◽  
Author(s):  
Ye Xie ◽  
Jia Yao ◽  
Weilin Jin ◽  
Longfei Ren ◽  
Xun Li
Keyword(s):  
Genetic Manipulation ◽  
Toxicity Testing ◽  
Basic Research ◽  
Drug Approval ◽  
Cell Types ◽  
Comprehensive Overview ◽  
Technological Advances ◽  
Degree Of Differentiation

Limited by the poor proliferation and restricted sources of adult hepatocytes, there is an urgent need to find substitutes for proliferation and cultivation of mature hepatocytes in vitro for use in disease treatment, drug approval, and toxicity testing. Hepatocyte-like cells (HLCs), which originate from undifferentiated stem cells or modified adult cells, are considered good candidates because of their advantages in terms of cell source and in vitro expansion ability. However, the majority of induced HLCs are in an immature state, and their degree of differentiation is heterogeneous, diminishing their usability in basic research and limiting their clinical application. Therefore, various methods have been developed to promote the maturation of HLCs, including chemical approaches, alteration of cell culture systems, and genetic manipulation, to meet the needs of in vivo transplantation and in vitro model establishment. This review proposes different cell types for the induction of HLCs, and provide a comprehensive overview of various techniques to promote the generation and maturation of HLCs in vitro.

scholarly journals PPM1D is a neuroblastoma oncogene and therapeutic target in childhood neural tumors

2020 ◽  
Author(s):  
Jelena Milosevic ◽  
Susanne Fransson ◽  
Miklos Gulyas ◽  
Gabriel Gallo-Oller ◽  
Thale K Olsen ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Genetic Manipulation ◽  
Clonal Evolution ◽  
Neuroblastoma Cell ◽  
Tumor Formation ◽  
T Cell Lymphomas ◽  
Wip1 Phosphatase ◽  
Bona Fide ◽  
Genome Scale

SUMMARYMajority of cancers harbor alterations of the tumor suppressor TP53. However, childhood cancers, including unfavorable neuroblastoma, often lack TP53 mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms.Here we show that p53-regulating PPM1D at chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. We identified that WIP1-phosphatase encoded by PPM1D, is activated by frequent segmental 17q-gain further accumulated during clonal evolution, gene-amplifications, gene-fusions or gain-of-function somatic and germline mutations. Pharmacological and genetic manipulation established WIP1 as a druggable target in neuroblastoma. Genome-scale CRISPR-Cas9 screening demonstrated PPM1D genetic dependency in TP53 wild-type neuroblastoma cell lines, and shRNA PPM1D knockdown significantly delayed in vivo tumor formation. Establishing a transgenic mouse model overexpressing PPM1D showed that these mice develop cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboring Notch1-mutations, Pten-deletions and p53-accumulation, adenocarcinomas and PHOX2B-expressing neuroblastomas establishing PPM1D as a bona fide oncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice proposing WIP1 as a therapeutic target in neural childhood tumors.

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