276 PRODUCTION OF TRANSGENIC MINI-PIG CELL LINES EXPRESSING HUMAN CYTOMEGALOVIRUS US2

2007 ◽  
Vol 19 (1) ◽  
pp. 254
Author(s):  
K. W. Park ◽  
J. Y. Yoo ◽  
K. M. Choi ◽  
S. P. Hong ◽  
G. S. Han ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cytomegalovirus ◽  
Recombinant Expression ◽  
Class I ◽  
Class I Mhc ◽  
Specific Lysis ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

Xenotransplantation has the potential to resolve the chronic shortage of donor organs if immunological barriers can be overcome. In particular, the initial type of rejection following xenotransplantation is acute cellular rejection by host CD8+ cytotoxic T lymphocyte (CTL) cells that react to the donor class I major histocompatibility complex (MHC). The human cytomegalovirus (HCMV) glycoprotein US2 specifically targets class I MHC heavy chains for dislocation from the endoplasmic reticulum (ER) membrane to the cytosol, where they are degraded by the proteasome. In this study, the recombinant expression vector pCX-US2 was stably transfected into mini-pig fetal fibroblasts by lipofection. The integration of US2 into the host genome was confirmed by PCR and Southern blot assay. The reduction of swine leukocyte antigen class I (SLA-I, MHC protein class I) by US2 was detected by flow cytometry analysis (FACS). FACS analysis of US2 clonal cell lines demonstrated substantial reductions in SLA surface expression. The decrease in the level of class I MHC expression for US2 clonal cell lines ranged from 22 to 34% relative to the non-transfected control. US2 clonal cell lines were also tested to determine if the resulting reduction in cell surface SLA would reduce in vitro cytotoxicity by CTL. The US2 clonal cell line demonstrated 5- to 6-fold reduction of specific lysis by primed CD8+ CTL. In conclusion, US2 can directly protect pig clonal cell lines from human CTL cells. These results indicate that the expression of US2 in pig cells may provide a new approach toward overcoming CTL-mediated immunity to xenotransplantation. This work was supported by the National Livestock Research Institute (6132-211-303-1).

193 HUMAN CYTOMEGALOVIRUS PROTEIN US11 DOWN-REGULATES THE EXPRESSION OF SWINE LEUKOCYTE ANTIGEN-I IN MINIPIG CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 176
Author(s):  
J. Y. Yoo ◽  
K. M. Choi ◽  
S. P. Hong ◽  
G. S. Han ◽  
E. J. Kim ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Cytomegalovirus ◽  
Cytotoxic T Lymphocyte ◽  
Early Stage ◽  
Swine Leukocyte Antigen ◽  
Leukocyte Antigen ◽  
Clonal Cell ◽  
Heavy Chains ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

The CD8+ cytotoxic T lymphocyte (CTL)-mediated immune response is important in porcine xenotransplantation, and pigs could be used as a good model for organ donor if these immunological barriers are overcome. Human cytomegalovirus (HCMV) encodes unique short (US) 11 gene, which interferes with cellular immune responses by inducing rapid degradation of newly synthesized heavy chains of major histocompatibility (MHC) class I. The destruction of heavy chains by US11 helps the virus to hide from recognition by cytotoxic T lymphocytes at early stage. In this study we demonstrated the inhibitory effect of US11 on the cytotoxicity of CTL cells by down-regulation of swine leukocyte antigen (SLA)-I expression. We established five US11 clonal cell lines by transfection into minipig fetal fibroblasts and confirmed the integration of US11 gene by PCR and Southern blot assay. The reduction of SLA-I, which was expressed on the cell surface, was also detected by flow cytometry assay. The level (14.6% to 21.2%) of SLA-1 expression in US11 clonal cell lines was decreased relative to that in the control. In the CTL assay, the rate of CD8+ T cell-mediated cytotoxicity was significantly reduced to 31.9 � 11.3%, compared to that of the control (81.4 � 5.3%; P < 0.01; n = 4). These results indicate that HCMV viral protein US11 can effectively suppress the presentation of SLA-I in pig fetal fibroblast cells. This work was supported by a grant (Code # 20070101034005) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

scholarly journals Plasticity of Hepatic Cell Differentiation: Bipotential Adult Mouse Liver Clonal Cell Lines Competent to Differentiate In Vitro and In Vivo

Stem Cells ◽  
2006 ◽  
Vol 24 (9) ◽  
pp. 2098-2109 ◽  
Author(s):  
Catherine Fougère-Deschatrette ◽  
Tereza Imaizumi-Scherrer ◽  
Hélène Strick-Marchand ◽  
Serban Morosan ◽  
Pierre Charneau ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Lines ◽  
Mouse Liver ◽  
Adult Mouse ◽  
Hepatic Cell ◽  
Clonal Cell ◽  
Clonal Cell Lines ◽  
Adult Mouse Liver

75 INHIBITION OF NK CELL CYTOTOXICITY IN CLONED MINI-PIGS EXPRESSING HUMAN LEUKOCYTE ANTIGEN-G1 (HLA-G1) GENE

2007 ◽  
Vol 19 (1) ◽  
pp. 155
Author(s):  
K. W. Park ◽  
G. S. Han ◽  
K. M. Choi ◽  
S. P. Hong ◽  
J. Y. Yoo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Control Group ◽  
Cell Cytotoxicity ◽  
Xenograft Rejection ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines ◽  
Mini Pigs

Human natural killer (NK) cell-mediated response plays an important role in xenograft rejection. In the case of pig-to-human xenotransplantation, it has been suggested that NK cells are involved in delayed-type rejection, which is characterized by pig endothelial cell (pEC) activation, direct lysis, and secretion of proinflammatory cytokines. NK cell activation can be a direct barrier to the potential use of pig organs for human xenograft transplantation. Therefore, it is important to suppress the NK cell activity on pig-to-human xenografts. Expression of HLA-G1 (non-classical major histocompatibility complex class I molecules) inhibits the cytotoxic activity of NK cells and has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity in pEC. In this study, we transfected the HLA-G1 gene into mini-pig fetal fibroblasts to produce 2 HLA-G1 clonal cell lines. These cell lines were used to produce cloned HLA-G1 transgenic mini-pigs by nuclear transfer (NT). The presence of the HLA-G1 gene in transgenic mini-pigs was confirmed by PCR. The expression of HLA-G1 was detected by flow cytometry-immunohistochemistry assay. Mini-pig fibroblasts derived from a 35-day-old cloned fetus also showed characteristics similar to those of HLA-G1 clonal cell lines. The expressed HLA-G1 significantly suppressed NK-mediated cell lysis, and the rate of NK 92MI cell cytotoxicity was reduced as compared to the control group (HLA-G1: 46.7 � 4.5%; control: 4.6 � 13.3%; P &lt; 0.05). In conclusion, transgenic cloned mini-pigs expressing HLA-G1 were produced by NT for the first time. It is expected that these mini-pigs could be used to overcome the NK cell-mediated rejection in xenotransplantation.

scholarly journals Osteoblasts are target cells for transformation in c-fos transgenic mice

1993 ◽  
Vol 122 (3) ◽  
pp. 685-701 ◽  
Author(s):  
AE Grigoriadis ◽  
K Schellander ◽  
ZQ Wang ◽  
EF Wagner
Keyword(s):  
Alkaline Phosphatase ◽  
Transgenic Mice ◽  
Cell Lines ◽  
Osteoblastic Cells ◽  
Target Cells ◽  
Type I ◽  
Class I Mhc ◽  
Clonal Cell ◽  
Fos Protein ◽  
Clonal Cell Lines

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone-forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c-fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype.

scholarly journals Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lucia Natalia Moro ◽  
Diego Luis Viale ◽  
Juan Ignacio Bastón ◽  
Victoria Arnold ◽  
Mariana Suvá ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Myostatin Gene ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

Abstract The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

scholarly journals Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

1988 ◽  
Vol 8 (4) ◽  
pp. 1664-1669 ◽  
Author(s):  
K M Karrer ◽  
M C Yao
Keyword(s):  
Cell Lines ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Host Cells ◽  
Rrna Genes ◽  
Major Constituent ◽  
Clonal Cell ◽  
Clonal Cell Lines

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

184 INHIBITION OF HUMAN COMPLEMENT-MEDIATED CYTOTOXICITY IN MINIPIG CELLS BY EXPRESSION OF hCD59

2008 ◽  
Vol 20 (1) ◽  
pp. 172
Author(s):  
K. W. Park ◽  
E. J. Kim ◽  
K. M. Choi ◽  
S. P. Hong ◽  
G. S. Han ◽  
...  
Keyword(s):  
Cell Lines ◽  
Membrane Attack Complex ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Control Group ◽  
Human Complement ◽  
Transgenic Pigs ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

Xenotransplantation of a pig organ to a human is a possible solution for the shortage of donor organs for transplantation. However, hyperacute rejection (HAR) due to natural antibodies (Nab) present major obstacle in pig-to-human xenotransplantation. To overcome this, much effort has been dedicated to preparing transgenic pigs that express human complement regulatory proteins (CRPs). One of CRPs, the human CD59 gene, can prevent the terminal polymerization of the membrane attack complex by complement. In this study, we investigated the inhibitory effect of hCD59 on complement-mediated cytotoxicity in hCD59-transfected minipig cells. To generate cell lines expressing hCD59, we transfected the hCD59 gene into minipig fetal fibroblasts and established seven transgenic clonal cell lines. The integration of hCD59 gene was confirmed by PCR and expression levels were measured by RT-PCR, fluorescence-activated cell sorting (FACS), Western blot, and immunohistochemistry. FACS analysis of hCD59 clonal cell lines demonstrated a substantial increment of hCD59 expression. The level (82% to 95%) of hCD59 protein expression was increased relative to that of the control. Human complement-mediated cytotoxicity was measured using a CytoTox96� (Promega Corp., Sydney, Australia) non-radioactive cytotoxicity (LDH) assay using normal minipig cells as a negative control. In the LDH release assay, human complement-mediated cytotoxicity was also significantly reduced to 39.6 � 17.8% in comparison to that of the control group (73.6 � 19.1%; P < 0.05; n = 6). These results indicate that the expression of hCD59 gene in minipig cells can efficiently control complement-mediated cytotoxicity.

Interleukin-6 stimulates cell proliferation of rat pituitary clonal cell lines in vitro

1995 ◽  
Vol 18 (2) ◽  
pp. 83-90 ◽  
Author(s):  
T. Sawada ◽  
Koji Koike ◽  
Y. Kanda ◽  
H. Ikegami ◽  
H. Jikihara ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Clonal Cell ◽  
Rat Pituitary ◽  
Clonal Cell Lines

scholarly journals Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro

2008 ◽  
Vol 237 (5) ◽  
pp. 1412-1423 ◽  
Author(s):  
J. Peltzer ◽  
L. Colman ◽  
J. Cebrian ◽  
H. Musa ◽  
M. Peckham ◽  
...  
Keyword(s):  
Cell Lines ◽  
Slow Muscle ◽  
Clonal Cell ◽  
Fast And Slow Muscle ◽  
Clonal Cell Lines
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