• Asian-Australasian Journal of Animal Sciences
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• v.18 no.2
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• pp.158-164
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• 2005

A practical approach was proposed to produce transgenic chimeric chickens using blastodermal cells (BCs). The chicken BCs were mechanically dissociated and transferred into the recipient eggs that had been exposed to 500 rads irradiation of$^{60}Co$ and windowed on the equatorial plane. Chimeric chickens were generated using two models: the crosses (MXL) from Black Minors (ii,EE,b/b) ♂${\times}$Barred Leghorns (ii,ee,B/-) ♀ as donors and White Leghorns (WL, II) as acceptors (Model 1), or the Black Heifengs (BH, ii,EE,bb) as donors and Hua-xing white (HW, II) as recipients (Model 2). The treated eggs were incubated in their original shells in normal conditions until hatching. Green fluorescent protein (GFP) gene was transferred into the BCs derived from MXL and BH via lipofectamine and the pEGFP-C1, and transfection efficiency into the BCs was examined under a fluorescent microscope. Potential transgenic chimeras were selected based on the proposed methods in this study. Using the fresh BCs, the best rate of phenotypic chimeras was 6.7% and 26.0% in model-1 groups, and model-2 groups, respectively. We also described the optimized conditions for transfection. Although 30% of the BCs transfected in vitro emitted green light under an inverted fluorescent microscope, no embryos injected with the transfected BCs expressed foreign GFP gene at 3-4 days.

• Korean Journal of Poultry Science
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• v.44 no.2
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• pp.103-111
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• 2017

The quail (Coturnix japonica) has been used as a model animal in many research fields and its application is still expanding in other fields. Compared to the chicken, the quail is quicker to reach sexually maturity, has short generation intervals, is easy to handle, requires less space and feed, and is sturdy. In addition, it produces many eggs and the research tools developed for chicken can be applied directly to quail or with some modifications. Due to recent advances in next-generation sequencing, abundant sequence data for the quail genome and transcripts have been generated. These sequence data are valuable sources for studying functional genomics using quail, which is one of the model animal used to investigate gene function and networks. Although there are some obstacles to be removed, the quail is the best optimized model to study the functional genomics of poultry. In many research fields, functional genomics study using the quail model will provide the best opportunity to understand the phenomena and principles of life. We review why, among many other birds, the quail is the best model for studying poultry functional genomics.

• Korean Journal of Poultry Science
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• v.46 no.1
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• pp.17-24
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• 2019

Chickens have been considered as well-defined animal bioreactor. The optimized ovalbumin promoter is essential for recombinant protein production in transgenic chicken. Here we try to compare the activity and identify the effect of estrogen on ovalbumin promoter according to each promoter length with estrogen response element (ERE) existence. We cloned two (2.8 and 5.5 kb) ovalbumin promoters that the 5.5 kb contained the ERE but the 2.8 kb did not, and these two promoters were cloned to pGL4.11 vector. Additionally, we constructed another pGL4.11 vector containing of the 4.4 kb (with ERE) ovalbumin promoter deleted with 1 kb between ERE region and the 2.8 kb promoter. For reporter assay, HeLa, MES-SA, LMH/2A, and cEF cells were transfected with all the pGL4.11 vectors. The comparative analysis showed that the mutated 4.4 kb promoter has more potent activity than the 2.8 and 5.5 kb promoters in HeLa, MES-SA, and LMH/2A cells. However, there is no significant difference in cEFs. Also, these cells transfected with the mutated 4.4 kb promoter were treated with the $17{\beta}$-estradiol (0~3,000 nM) and HeLa, MES-SA, and LMH/2A cells showed estrogen responsibilities, but cEFs did not. Besides, the mutated 4.4 kb promoter has still higher activity than the 2.8 and 5.5 kb promoter, and there is no transcriptional induction effect in 2.8 kb promoter at 500 nM estrogen that is blood concentration of laying hens. Hence our study strongly suggested that the mutated 4.4 kb promoter is considered as one of the most efficient length for generating transgenic chicken.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2006.11a
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• pp.43-58
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• 2006

As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.207-211
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• 2015

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken ${\beta}-actin$ promoter-driven firefly improved luciferase cDNA (${\beta}-act/luc^+$) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (${\beta}-act/luc^+$) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.145-145
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.145-145
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• 2005

• Korean Journal of Poultry Science
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• v.28 no.2
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• pp.155-163
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• 2001

Gene and cell transfer technique will serve as a powerful tool for the genetic improvement of the poultry and to yield useful products. For avian transgenesis, Japanese quail may serve as an excellent animal model because of its small body size and fast growth rate. Recent progress was described on the manipulation of quail embryos such as the introduction of foreign genes and cells, and the subsequent culturing of the manipulated embryos yielding hatchlings. Intraspecific donor-derived offspring have been available in quail, however, further investigation will be required to obtain interspecific offspring with the aim of rescuing endangered species. Trans genesis will also be useful for improving the profitability and quality of poultry stocks and for developing stocks with novel uses. Considerable progress should soon be made toward the production of transgenic poultry. The key feature of the procedure described here is that embryos are initially taken out from the shell for ease of manipulation and then placed back in culture in addition to various operations midway during culture.

• Animal Bioscience
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• v.36 no.2_spc
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• pp.333-338
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• 2023

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2005.11a
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• pp.66-67
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• 2005

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGC) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 day of incubation, and the gPGC were cultured in vitro until colony formed. After 7-10 days in cultured gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of thistype will serve as an important reference for germ cell biology and transgenic research.