91 Expression Pattern of Neuron-Specific Red Fluorescence Protein in Nervous System of Transgenic Dog with Human Synapsin I Promoter

2018 ◽  
Vol 30 (1) ◽  
pp. 185
Author(s):  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
E. M. N. Setyawan ◽  
S. H. Lee ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Neurodegenerative Disorders ◽  
Peripheral Nerves ◽  
Synapsin I ◽  
Neural Cells ◽  
Tissue Samples ◽  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein

Neurodegenerative disorders, such as Alzheimer’s disease, affect neurons in large areas within the central nervous system. The selective expression of disease-causing and therapeutic genes in susceptible regions and cell types is critical to the generation of animal models. In our previous studies, we reported production of a transgenic dog by nuclear transfer using donor cells that had been stably transfected by vector containing the red fluorescence protein (RFP) and human synapsin I (SynI) promoter, a neuron-specific promoter. Here, we evaluated whether RFP expression in transgenic dog with SynI (SynI dog) had high neuronal specificity and strong transcriptional activity. For confirmation of neuron-specific RFP expression, tissue samples were obtained from a killed 4-year-old SynI dog. Quantification of RFP in heart, kidney, liver, lung, spleen, cerebrum, cerebrum, cerebellum, midbrain, hippocampus, peripheral nerves, skin, and spinal cord of SynI dog was analysed using ELISA (Cell Biolabs, San Diego, CA, USA). Moreover, the distribution of RFP activity in neural system of the SynI dog was determined by immunohistochemistry. The first antibody, rabbit polyclonal RFP antibody (1:200, ab62341, Abcam, Cambridge, MA, USA) was applied, and antibody labelling was visualised by incubation with avidin-biotinylated horseradish peroxidase complex (1:1,000; ABC Elite, Vector Laboratories, Burlingame, CA, USA). In the 4-year-old SynI dog, RFP was observed only in neuronal tissues including brain, spinal cord, and peripheral nerves, but was not detected in non-neuronal tissue such as heart, kidney, and skin. In addition, robust RFP expression was observed in the neurons of the peripheral nerve, spinal cord, and prefrontal cortex. In the hippocampus and cerebellum, the RFP-expressing cells appeared heterogeneous in hippocampus neurons and purkinje cells. In conclusion, we report that human SynI promoter is functional in neural cells of dogs. A neural specific-transgene expressed dog could be applied as a research tool in the study of neurodegenerative disorders. This research was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), NRF (#2016R1D1A1B03932198), and Research Institute for Veterinary Science, the BK21 plus program.

32 NEURON-SPECIFIC EXPRESSION OF THE RED FLUORESCENCE PROTEIN IN CLONED DOGS

2012 ◽  
Vol 24 (1) ◽  
pp. 128 ◽  
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
G. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Degenerative Disease ◽  
Neural Cells ◽  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
The Brain ◽  
Synapsin 1

The pathogenesis of neuronal degenerative disease as Alzheimer's disease (AD) has been a subject of intensive research for the last few decades worldwide. But despite such effort, treatment or preventive measures for AD have so far made no breakthrough. One of the contributing factors that hindered the progress of research is the lack of appropriate AD models. Mouse models have limitations for AD research because the irreconcilable species gap between the rodent and human has impeded the research itself as well as the application of the findings from the rodent studies to human cases. As an alternative, here we performed a preliminary study to develop novel neuronal degenerative disease models using a canine transgenic somatic cell nuclear transfer (SCNT) technology. The aim of this study is to produce a transgenic dog that expresses neuron-specific transgene in the brain by SCNT. In this study, we chose human synapsin 1 promoter as primarily neuron selective, driving the red fluorescent protein transgene. For SCNT, synapsin 1-red fluorescence protein (SYN1-RFP) was introduced into female beagle adipose-derived stem cell via lentiviral vector infection. The SYN1-RFP cells were injected into enucleated in vivo-matured dog oocytes and fused by electric stimulation. The fused couplets (80/94, 85.1%) were chemically activated and transferred into the uterine tube of 5 naturally oestrus-synchronized surrogates. Three of them (60%) maintained pregnancy and subsequently gave birth to 3 cloned pups (SYN1-RFP A, SYN1-RFP B, SYN1-RFP C) by natural delivery or cesarean section. Birth weights of the offspring ranged from 120 to 280 g and SYN1-RFP C is still alive, healthy and does not show any abnormalities. The microsatellite analysis shows that all SYN1-RFP puppies originated from the SYN1-RFP cells used in SCNT and mitochondrial DNA analysis shows that the puppies had been derived from the oocyte donors. In order to investigate the result in multiple transgene insertions, SYN-RFP puppies were screened by Southern blot analysis using DNA extracted from skin biopsies. Transgene copy number was estimated by Southern blot analysis. The SYN-RFP A and B that died at 3 days after birth had approximately 5 and 2 copies of the transgene integrated, respectively, whereas the alive SYN-RFP C has 1 copy. SYN-RFP B was particular in that it did not express RFP in the entire body, but samples collected postmortem showed expression of the RFP transgene under the human synapsin 1 promoter in neural cells in the brain of SYN-RFP B. In conclusion, we report here that (1) the human synapsin promoter is functional in neural cells of dog brain and (2) a neural-specific-transgene-expressed dog was generated for the first time by transgenic SCNT technique. Furthermore, the SYN-RFP dog has great potential to understand the function of a neuronal degenerative disease model dog. This study was supported by MKE (Grant # 10033839-2011-13), RNL Bio, IPET and TS Corporation.

302 CONFIRMATION OF GERM-LINE TRANSMISSION OF RED FLUORESCENCE PROTEIN TRANSGENIC MALE CAT

2009 ◽  
Vol 21 (1) ◽  
pp. 248 ◽  
Author(s):  
S. J. Cho ◽  
Y. S. Lee ◽  
E. G. Choi ◽  
J. I. Bang ◽  
X. F. Yu ◽  
...  
Keyword(s):  
Germ Line ◽  
Basic Research ◽  
Transgenic Animals ◽  
Pcr Analysis ◽  
Parental Line ◽  
Tissue Samples ◽  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Natural Mating

Production of transgenic animals is highly desirable for biotechnology and basic research. Therefore, a method of producing genetically modified cats through genetic engineering is important for generating biomedical models of human diseases. We investigate reproductive ability of red fluorescence protein (RFP) transgenic cloned male cat (RFP TG cat) in natural mating with domestic female cat. One domestic female cat in natural mating with RFP TG cat delivered 6 kittens of which 3 (2 female and 1 male) showed RFP expression of parental line. Among the 3 RFP expressing kittens, 1 died at day 5 and were subjected to PCR analysis for confirmation of RFP gene expression. Red fluorescence protein gene was detected in tissue samples including liver, muscle, brain, large intestine, bladder, spleen, sexual organ, small intestine, kidney, heart, stomach, pancreas, lungs, and skin of the kitten. These results indicated that RFP transgenic cloned male cat have normal reproductive fertility, and the stability of transmission of the RFP transgene through the germ line and its normal expression in offspring. Supported by KOSEF (M10525010001-05N2501-00110). S. J. Cho, Y. S. Lee, E. G. Choi, and J. I. Bang were supported by a scholarship from the Post BK21 Program, the Ministry of Education, Science and Technology, Korea.

scholarly journals The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

2016 ◽  
Vol 7 ◽  
Author(s):  
Mariamichela Lanzilli ◽  
Giuliana Donadio ◽  
Roberta Addevico ◽  
Anella Saggese ◽  
Giuseppina Cangiano ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein

56 ESTABLISHMENT OF TRANSGENIC RED FLUORESCENCE PROTEIN (RFP) CLONE DOGS THROUGH A STABLE TRANSMISSION OF RFP GENE TO NEXT GENERATION

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
G. A. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Second Generation ◽  
Pcr Analysis ◽  
Next Generation ◽  
Wild Type ◽  
Red Fluorescence ◽  
Reproductive Ability ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Regulated Gene Expression ◽  
Stable Transmission

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.

Confirmation of Germ-Line Transmission in the Red Fluorescence Protein (RFP) Transgenic Cloned Male Cat

2010 ◽  
Vol 12 (6) ◽  
pp. 739-747 ◽  
Author(s):  
Su-Jin Cho ◽  
Young S. Lee ◽  
Jae-Ik Lee ◽  
Jae-Il Bang ◽  
Jing Yang ◽  
...  
Keyword(s):  
Germ Line ◽  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein ◽  
Germ Line Transmission

scholarly journals Neuromonitoring

2020 ◽  
pp. S35-S39
Author(s):  
M Dlamini
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Surgical Approach ◽  
Peripheral Nerves ◽  
Conscious State ◽  
Awake Patient ◽  
Permanent Damage ◽  
Complex Interactions ◽  
The Brain ◽  
Different Parts

Neuromonitoring is used during surgery to assess the functional integrity of the brain, brain stem, spinal cord, or peripheral nerves. The aim of monitoring is to prevent permanent damage by early intervention when changes are detected in the monitor. Neuromonitoring is also used to map areas of the nervous system in order to guide management in some cases. The best neuromonitor remains the awake patient. In the conscious state, the function of individual parts of the nervous system and the complex interactions of its different parts can be assessed more accurately. However, most surgical procedures involving the nervous system require general anaesthesia. Procedures that require neuromonitoring can have changes in their monitored parameters corrected by modifying the surgical approach or by having the anaesthesiologist manipulate the parameters under their control. An ideal neuromonitor would be one that is specific for the parameter of interest, and gives reliable, reproducible, or continuous results.

scholarly journals Immunolocalization of a neuronal growth-dependent membrane glycoprotein.

1985 ◽  
Vol 101 (5) ◽  
pp. 1990-1998 ◽  
Author(s):  
I Wallis ◽  
L Ellis ◽  
K Suh ◽  
K H Pfenninger
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Growth Cone ◽  
Cortical Neurons ◽  
Axonal Growth ◽  
Staining Intensity ◽  
Olfactory Nerve ◽  
Membrane Glycoprotein ◽  
Neural Cells ◽  
Developing Spinal Cord

Monoclonal antibody (mAb) 5B4 recognizes in the rat a large, developmentally regulated membrane glycoprotein. The larger form of this antigen (185-255 kD) occurs in the developing nervous system and is present in membranes of nerve growth cones, as determined by analysis of a growth cone particle fraction. An immunochemical characterization of this antigen and of a smaller form (140 kD), sparsely present in the mature nervous system, has been described (Ellis, L., I. Wallis, E. Abreu, and K. H. Pfenninger, 1985, J. Cell. Biol., 101:1977-1989). The present paper reports on the localization by immunofluorescence of 5B4 antigen in cultured cortical neurons, developing spinal cord, and the mature olfactory system. In culture, mAb 5B4 stains only neurons; it is sparsely present in neurons at the onset of sprouting while, during sprouting, it appears to be concentrated at the growth cone and in regions of the perikaryon. In the developing spinal cord, 5B4 labeling is faintly detectable on embryonic day 11 but is intense on fetal day 13. At this stage, the fluorescence is observed in regions of the cord where axonal growth is occurring, while areas composed of dividing or migrating neural cells are nonfluorescent. With maturation of the spinal cord, this basic pattern of fluorescence persists initially, but the staining intensity decreases dramatically. In the adult, faint fluorescence is detectable only in gray matter, presumably indicating the presence of the 140 kD rather than the fetal antigen. The only known structure of the adult mammalian nervous system where axonal growth normally occurs is the olfactory nerve. mAb 5B4 intensely stains a variable proportion of olfactory axons in the mucosa as well as in the olfactory bulb. Based on both immunochemical and immunofluorescence data, the 5B4 antigen of 185-255 kD is associated specifically with growing neurons, i.e., neurons that are generating neurites.

Coral red fluorescence protein as genetic modified baculovirus tracer

2005 ◽  
Vol 119 (3) ◽  
pp. 255-259 ◽  
Author(s):  
Tzyy-Rong Jinn ◽  
Suey-Sheng Kao ◽  
Jason T.C. Tzen ◽  
Tzong-Yuan Wu
Keyword(s):  
Red Fluorescence ◽  
Fluorescence Protein ◽  
Red Fluorescence Protein

scholarly journals Studies on the Regulatory Roles and Related Mechanisms of lncRNAs in the Nervous System

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zijian Zhou ◽  
Dake Qi ◽  
Quan Gan ◽  
Fang Wang ◽  
Bengang Qin ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Nerve Injury ◽  
Peripheral Nerves ◽  
Growth And Development ◽  
Noncoding Rnas ◽  
Regulatory Function ◽  
Full Understanding ◽  
Cellular Processes ◽  
Physiological Processes

Long noncoding RNAs (lncRNAs) have attracted extensive attention due to their regulatory role in various cellular processes. Emerging studies have indicated that lncRNAs are expressed to varying degrees after the growth and development of the nervous system as well as injury and degeneration, thus affecting various physiological processes of the nervous system. In this review, we have compiled various reported lncRNAs related to the growth and development of central and peripheral nerves and pathophysiology (including advanced nerve centers, spinal cord, and peripheral nervous system) and explained how these lncRNAs play regulatory roles through their interactions with target-coding genes. We believe that a full understanding of the regulatory function of lncRNAs in the nervous system will contribute to understand the molecular mechanism of changes after nerve injury and will contribute to discover new diagnostic markers and therapeutic targets for nerve injury diseases.

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