scholarly journals Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0194721 ◽  
Author(s):  
Mo Sun Kwon ◽  
Bon Chul Koo ◽  
Dohyang Kim ◽  
Yu Hwa Nam ◽  
Xiang-Shun Cui ◽  
...  
Keyword(s):  
Transgenic Chicken ◽  
Human Erythropoietin ◽  
Specific Manner ◽  
Transgenic Chickens ◽  
Chicken Eggs

scholarly journals Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 38
Author(s):  
Takehiro Mukae ◽  
Sho Okumura ◽  
Takuma Watanobe ◽  
Kyoko Yoshii ◽  
Takahiro Tagami ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Capacity ◽  
Specific Binding ◽  
Egg White ◽  
Peptide Sequence ◽  
Efficient Production ◽  
Antigen Binding ◽  
Transgenic Chicken ◽  
Bioreactor System ◽  
Transgenic Chickens

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4–1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.

scholarly journals High-Level Expression of Single-Chain Fv-Fc Fusion Protein in Serum and Egg White of Genetically Manipulated Chickens by Using a Retroviral Vector

2005 ◽  
Vol 79 (17) ◽  
pp. 10864-10874 ◽  
Author(s):  
Masamichi Kamihira ◽  
Ken-ichiro Ono ◽  
Kazuhisa Esaka ◽  
Ken-ichi Nishijima ◽  
Ryoko Kigaku ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Leukemia Virus ◽  
Retroviral Vector ◽  
Single Chain ◽  
Transgenic Chicken ◽  
Chicken Embryos ◽  
Single Chain Fv ◽  
High Level ◽  
Transgenic Chickens ◽  
Transgenic Progeny

ABSTRACT We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.

336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

2013 ◽  
Vol 25 (1) ◽  
pp. 315
Author(s):  
B. Koo ◽  
M. Kwon ◽  
J. Roh ◽  
J. Kim ◽  
T. Kim
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Inducible Expression ◽  
Gfp Expression ◽  
Transgenic Chicken ◽  
Inducible Expression System ◽  
Green Fluorescent ◽  
Transgenic Chickens

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.

scholarly journals Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

2016 ◽  
Vol 8 (10) ◽  
pp. 63
Author(s):  
Saisai Wang ◽  
Yali Wang ◽  
Dan Shen ◽  
Li Zhang ◽  
Songlei Xue ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Transfer Efficiency ◽  
Transgenic Chicken ◽  
Simple Method ◽  
Gene Transfer Efficiency ◽  
Transgenic Chickens

<p>Transposon mediated transfection is a promising, safe, and convenient way to generate transgenic chicken compared with virus-mediated technology and the in vitro modification of primordial germ cells (PGCs). To establish a simple method for in vivo transfection of chicken PGCs, we applied four different transposon systems (PB, SB, Tol2, and ZB) to investigate the gene transfer efficiency of chicken gonads via direct injection of a mixture of transposon and transposase plasmids and transfection reagent (polyethylenimine, PEI) into the subgerminal cavity of Hamburger and Hamilton stage 2-3 chick embryos. We also compared the effect of the amount of plasmids injected on the gene transfer efficiency of chicken gonads. We found that over 70% of the gonads were green fluorescent protein (GFP)-positive across all four transposon groups, and that the proportion of GFP-positive gonads was not significantly different between different transposons. Some GFP positive cells in gonads were confirmed as germ cells by co-labeling with the germ cell specific antibody. We also found that the proportions of GFP-positive gonads decreased significantly with a decrease of plasmid dose from 100 ng to 20 or 50 ng. Here we revealed that a combination of transposons with PEI is a simple and efficient method for gene transfer into chicken gonads and able to transfect PGCs in vivo that could be used for the production of transgenic chickens.</p>

Production of transgenic chickens constitutively expressing human erythropoietin (hEPO): Problems with uncontrollable overexpression of hEPO gene

2017 ◽  
Vol 22 (1) ◽  
pp. 22-29 ◽  
Author(s):  
Bon Chul Koo ◽  
Mo Sun Kwon ◽  
Dohyang Kim ◽  
Sang A. Kim ◽  
Nam-Hyung Kim ◽  
...  
Keyword(s):  
Human Erythropoietin ◽  
Transgenic Chickens

scholarly journals In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

2021 ◽  
Vol 9 (1) ◽  
pp. 164
Author(s):  
Arjun Challagulla ◽  
Kristie A. Jenkins ◽  
Terri E. O’Neil ◽  
Shunning Shi ◽  
Kirsten R. Morris ◽  
...  
Keyword(s):  
Chicken Embryo Fibroblast ◽  
Marek's Disease ◽  
Recombinant Vaccines ◽  
Marek’S Disease ◽  
Transgenic Chicken ◽  
Guide Rnas ◽  
Specific Virus ◽  
Transgenic Chickens ◽  
Important Disease

Marek’s disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Primordial germ cells (PGCs) as a tool for creating transgenic chickens

2012 ◽  
Vol 15 (1) ◽  
pp. 181-188 ◽  
Author(s):  
L. Chojnacka-Puchta ◽  
K. Kasperczyk ◽  
G. Płucienniczak ◽  
D. Sawicka ◽  
M. Bednarczyk
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Heterologous Proteins ◽  
Transgenic Chicken ◽  
Migration Pathway ◽  
Pharmaceutical Proteins ◽  
Alternative Approach ◽  
Recent Developments ◽  
Transgenic Chickens

Primordial germ cells (PGCs) as a tool for creating transgenic chickens The transgenic chicken has great potential as a bioreactor for the production of valuable pharmaceutical proteins, notably in the oviduct/egg. Whereas conventional transgenic approaches have significant limitations in this species, an alternative approach employing primordial germ cells (PGCs), the progenitor cells to ova and spermatozoa, has now been successfully applied to the insertion of exogenous genes into birds. Recent developments in manipulating avian embryos make it possible to produce germline chimeras derived from transferred PGCs. In this review we describe the migration pathway of chicken PGCs during early development. We then summarize different methods for the isolation of PGCs and the diversity of techniques used to introduce genes into these cells. Finally, we describe an in vitro assay for testing tissue-specific vectors designed to express heterologous proteins in transgenic chickens.

Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens

2009 ◽  
Vol 19 (3) ◽  
pp. 437-447 ◽  
Author(s):  
Bon Chul Koo ◽  
Mo Sun Kwon ◽  
Hyuna Lee ◽  
Minjee Kim ◽  
Dohyang Kim ◽  
...  
Keyword(s):  
Human Erythropoietin ◽  
Transgenic Chickens

375 PRODUCTION OF GERMLINE TRANSGENIC CHICKENS USING MURINE LEUKEMIA VIRUS-BASED RETROVIRUS VECTOR SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 295
Author(s):  
B. C. Koo ◽  
M. S. Kwon ◽  
B. R. Choi ◽  
W. Chang ◽  
I. Jeon ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Green Fluorescence Protein ◽  
The Body ◽  
Vector System ◽  
Whole Body ◽  
Green Fluorescence ◽  
Transgenic Chicken ◽  
Egfp Gene ◽  
Retrovirus Vector ◽  
Transgenic Chickens

We report here successful generation of germline transgenic chickens expressing the enhanced green fluorescence protein (EGFP) gene throughout whole bodies. The founder chickens were produced by injecting replication-defective recombinant retroviruses encapsulated with the vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped retrovirus vector system beneath the blastoderm of non-incubated chicken embryos (stage X). Of 129 injected eggs, 13 chickens hatched. These 13 chickens were analyzed at 6-16 weeks post-hatching and all were found to emit green fluorescence in at least one part of the body. One cock of the 13 Go chimeric founder cockerels was mated with several non-transgenic hens, and one of 102 G1 siblings was found to emit a green fluorescent signal in its whole body. Successful germline transmission of the EGFP transgene was further confirmed in the G2 generation chickens: Crossing of the G1 cock and several non-transgenic hens resulted in about 50% (18/37) of the G2 cockerels being transgenic. The results of this study would be very helpful in establishing a useful transgenic chicken model system for generation of transgenic chickens as bioreactors producing therapeutic proteins, as well as for studies on embryo development.

304 PRODUCTION OF TRANSGENIC CHICKENS EXPRESSING HUMAN ERYTHROPOIETIN IN A TETRACYCLINE-INDUCIBLE MANNER

2009 ◽  
Vol 21 (1) ◽  
pp. 249
Author(s):  
B. Koo ◽  
M. Kwon ◽  
J. Roh ◽  
H. Lee ◽  
N. Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chicken Embryo ◽  
Hepatitis Virus ◽  
Expression System ◽  
Development Program ◽  
Transgenic Animals ◽  
Republic Of Korea ◽  
Efficient Production ◽  
Human Erythropoietin ◽  
Transgenic Chickens

The use of livestock animals as bioreactors to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to agricultural improvement and medical advancement. We report here the production of transgenic chickens that produce human erythropoietin (hEPO) using replication-defective Moloney murine leukemia virus-based vectors. Because it is well known that constitutive overexpression of some cytokine genes in the transgenic animals may cause serious physiological disturbances, the vectors were designed to express in the presence of tetracycline. In addition, we introduced woodchuck hepatitis virus posttranscriptional regulatory element sequence at 3′ end of hEPO gene to boost the gene expression under the inducible condition. Approximately 5 μL of vector virus solution concentrated as much as 109 cfu mL–1 was injected beneath the blastoderm of non-incubated chicken embryo (stage X). Out of 596 injected eggs, 36 chicks hatched after 21 days of incubation and 23 hatched chicks were found to express vector-encoded hG-GSF gene when fed with doxycycline. Quantitative analysis of the blood samples taken from some Go transgenic chickens resulted in more than 300 IU mL–1 of hEPO in the blood. These transgenic chickens have not exhibited any physiological abnormalities; therefore, it is possible that this controllable gene expression system may be useful in minimizing detrimental side effects when used to produce other transgenic animals. The biological activity of the recombinant hEPO was comparable to its commercially derived Escherichia coli counterpart. The significance of this work stems from the fact that it is the first successful report on the production of transgenic chickens expressing the hEPO gene. This approach can be employed to create a useful transgenic model system for further studies on the chicken embryo development and the efficient production of transgenic chickens as bioreactors. This work was supported by: The BioGreen 21 Program of the Rural Development Administration, Republic of Korea; The SRC/ERC program of MOST/KOSEF (grant no. R11-2002-100-04005-0); The 2006–2011 Technology Development Program for Agriculture and Forestry (TDPAF), Ministry of Agriculture and Forestry, Republic of Korea.

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