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.

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.

330 REDUCED HYPERACUTE REJECTION BY TRIPLE TRANSGENIC EXPRESSION OF HUMAN COMPLEMENT REGULATORY FACTORS (hDAF and hCD59) AND H-TRANSFERASE

2011 ◽  
Vol 23 (1) ◽  
pp. 261
Author(s):  
Y. H. Jeong ◽  
G. H. Jang ◽  
I. S. Hwang ◽  
C. H. Park ◽  
H. J. Lee ◽  
...  
Keyword(s):  
Human Serum ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Vector ◽  
Inhibitory Effect ◽  
Hyperacute Rejection ◽  
Stable Cell Line ◽  
Control Group ◽  
Human Complement ◽  
Transgenic Cell

The present study was conducted to establish a porcine transgenic cell line with human CRPs and HT genes, focused on hyperacute rejection (HAR) considering clinical xenotransplantation as alternative sources of human organs. As a first step towards establishing the stable cell line, the cDNA for 3 genes encoding human DAF, CD59, and H-transferase were cloned and sequenced. A tricistronic expression vector was constructed with the aid of 2 IRES elements (pCMV-hDAF_IRES-hHT_IRES-hCD59). The CMV-based expression vector was then introduced into miniature pig ear fibroblast cells by electroporation. Reverse transcription PCR analysis revealed that cell lines stably expressing human transgene-specific transcripts were established. The inhibitory effect of immune response in the established transgenic cell lines was measured by human serum-mediated cytolysis assay, as measured by ELISA. Under the assay conditions (based on human serum from 10 to 50%), the transgenic cell group showed significantly greater survival rate under various serum concentrations than did the nontransgenic cell control group. Moreover, the transgenic cell lines used as nuclear donors for a subsequent NT experiment were confirmed to be expressing their transgene transcripts in vitro developed preimplantation stage embryos. These results indicated that the established cell lines with human transgenes might have an inhibitory effect against lysis by human complement. It is possible that these transgenic cells could serve as nuclear donors to produce transgenic cloned pigs for xenotransplantation.

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.

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.

ESTABLISHMENT AND CHARACTERIZATION OF CLONAL CELL LINES FROM THE VAGINA OF p53-DEFICIENT YOUNG MICE

2002 ◽  
Vol 38 (10) ◽  
pp. 547 ◽  
Author(s):  
KAYO TANAHASHI ◽  
SHINOBU SHIBAHARA ◽  
MINAKO OGAWA ◽  
MAKOTO HANAZONO ◽  
SHINICHI AIZAWA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Clonal Cell ◽  
Clonal Cell Lines ◽  
Young Mice

Implantation of c-mycERTAMImmortalized Human Mesencephalic-Derived Clonal Cell Lines Ameliorates Behavior Dysfunction in a Rat Model of Parkinson’s Disease

2009 ◽  
Vol 18 (2) ◽  
pp. 307-320 ◽  
Author(s):  
Erik A. Miljan ◽  
Susan J. Hines ◽  
Priyadarshini Pande ◽  
Randolph L. Corteling ◽  
Caroline Hicks ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Rat Model ◽  
Clonal Cell ◽  
Clonal Cell Lines
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