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.

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.

scholarly journals Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2566-2571 ◽  
Author(s):  
FM Stewart ◽  
RB Crittenden ◽  
PA Lowry ◽  
S Pearson-White ◽  
PJ Quesenberry
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Normal Male ◽  
Normal Donor ◽  
Long Term Stability ◽  
Donor Bone Marrow ◽  
Male Donor ◽  
Marrow Cells

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

scholarly journals Mutational analysis of the CDKN2 (MTS1/p16ink4A) gene in primary B-cell lymphomas

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2724-2731 ◽  
Author(s):  
T Uchida ◽  
T Watanabe ◽  
T Kinoshita ◽  
T Murate ◽  
H Saito ◽  
...  
Keyword(s):  
B Cell ◽  
Missense Mutation ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Lymphocytic Leukemia ◽  
Sscp Analysis ◽  
Putative Tumor Suppressor Gene

Abstract The CDKN2 gene located on chromosome 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16. This gene is a putative tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the CDKN2 gene to evaluate its alterations in 52 primary specimens of non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southern blot analysis, polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing. By Southern blot analysis, we showed homozygous deletion of the CDKN2 gene in 3 of 42 patients with B-NHL (7.1%). After screening by PCR-SSCP analysis, direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp deletion arising at codon 49 (nucleotides 163 to 175) in patients with B-NHL (3 of 42, 7.1%). In the patient carrying the missense mutation, hemizygous deletion of the CDKN2 gene was also suspected. In this study, we detected alterations in CDKN2 in 6 of 42 patients (14.3%) with B-NHL and in none of 10 patients with B-CLL. Our results suggest that the CDKN2 alterations contribute in tumorigenesis in some patients with B-NHL.

Analysis of HLA-B35 variants andB35 haplotypes by isoelectric focusing and Southern blot analysis

1989 ◽  
Vol 30 (1) ◽  
pp. 63-65 ◽  
Author(s):  
Erich Lederer ◽  
Elfriede Nößner ◽  
Rudolf Wank ◽  
Dolores J. Schendel
Keyword(s):  
Southern Blot ◽  
Isoelectric Focusing ◽  
Blot Analysis ◽  
Southern Blot Analysis
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