• 한국가금학회지
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• 제26권2호
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• pp.109-118
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• 1999

Hen's eggs have been regarded as one of the best animal bioreactors to produce biologically active peptides originated from many organisms including human. Despite the last decade's efforts to produce transgenic chicken for any commercial purposes, the results so far reported are very disappointing, indicating that hen's eggs are very difficult to crack for transgenesis. Comparatively large female gamete with enormous amount of yolk may be one of the major obstacles in achieving a similar feat to those of other vertebrate species including mouse, sheep, fish and frog. The delay or less efficiency evidenced may instruct to try an alternative way of gens transfer into chicken egg. Sperm-mediated gene transfer is one of them, and may require a great deal of understanding of mechanisms involved in early fertilization and embryonic development. In other animals where the technique was successful, basic mechanisms have been well studied and established only by painstaking efforts for decades. This paper discusses the accumulated knowledge on early fertilization mechanism in the chicken and how can this information be utilitzed to find the alternative gene transfer in making transgenic chicken.

• Bulletin of the Korean Chemical Society
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• 제33권5호
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• pp.1536-1540
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• 2012

Chicken primordial germ cells (cPGCs) are founder germ cells in embryonic stage of development that eventually give rise to sperms or oocytes. Currently cPGCs are only known cells enabling germline transmission in chicken and their cultivation protocols were recently established. Although genome modifications of chickens are now theoretically possible using cPGCs, there are still several hurdles to overcome to practically use cPGCs as mediators for chicken transgenesis. First, efficiency of gene delivery into cPGCs remains low with current methods. Second, there aregene silencing mechanisms against the expression of foreign genes in cPGCs. In this study, we successfully increased the efficiency of gene delivery in cPGCs by taking advantage of the TTAA-specific $piggybac$ transposon system. Moreover, a pipette-type electroporator significantly enhanced transfection efficiency up to 5-fold compared withcuvette-type methods. Taken together, the technological advances in our study will provide practical benefits for the application to fulfill genetic modifications of chicken genome.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2009년도 춘계학술대회
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• pp.4-5
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• 2009

• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1227-1231
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• 2002

A powerful tool for chicken transgenesis could be established by employing a germline chimera production through primordial germ cell transplantation. This study was conducted to examine whether foreign gene-transfected gonadal primordial germ cells (gPGCs) have a migration activity into the gonad after transfer to recipient embryos. In Experiment 1, gPGCs of Korean Ogol Chicken were retrieved from 5.5-day-old embryos and subsequently transferred to the dorsal aorta of 2.5-day-old White Leghorn embryos after being labeled with PKH26 fluorescent dye. To confirm migration activity after transplantation, recipient embryos were sacrificed and examined on 3 days after transfer. Sex determination was concomitantly undertaken to examine whether sex of recipient embryos could affect the migration activity of gPGCs. All of embryonic gonads examined showed positive signals with PKH26 fluorescence and W-chromosome specific band by polymerase chain reaction (PCR) was detected in male embryos when gPGCs with ZW chromosome were transferred to recipient embryos. In Experiment 2, retrieved gPGCs were transfected with LacZ gene-containing cytomegalovirus promoter ($pCMV{\beta}$) by electroporation and subsequently transferred to recipient embryos. LacZ gene expression was identified in the gonads of 6 or 10-day-old recipient embryos and hatched-chicks. A total of 20 embryos and 12 hatched-chicks were examined and 11 of them (10 embryos and one hatched chicken; 11/32=34.4%) expressed $\beta$-galactosidase, a marker substance of LacZ gene. The results of this study demonstrated that foreign gene-transfected gPGCs can migrate and settle down into the gonad after being transferred into the blood vessel of the recipient embryos. This established technique will contribute to developing a peer biotechnology for transgenic chicken.

• Asian-Australasian Journal of Animal Sciences
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• 제17권12호
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• pp.1652-1656
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• 2004

The aim of this study was to obtain mature ova or embryos at a single cell stage, which can be used in avian transgenesis and nuclear transfer through multiple ovulations, in vitro fertilization and culture. Chicken anterior pituitary extract (CAPE) or acetone-dried chicken anterior pituitary extract (ACAPE) was used to induce multiple ovulations in hens pretreated with pregnant mare' serum gonadotrophin (PMSG). In vitro fertilization of the multiple ovulated ova was performed by inseminating sperm onto the germinal disks in m-Ringer' solution and incubating the ova at 41$^{\circ}C$, 5% $CO_2$ for 10 h in DME-F12 medium containing 20% liquid albumen. The in vitro fertilization process was observed using an environmental scanning electron microscope. When normal laying hens (white Leghorn) were administered daily with PMSG (100 IU), egg laying ceased in most hens within 3 to 8 days. Ovulation began to occur about 7.5 h after injection of CAPE and ACAPE. The number of ovulated ova was 1.00${\pm}$0.00, 2.33${\pm}$0.52 and 2.20${\pm}$0.45, respectively, after receiving 100, 200 and 300 mg CAPE. The number of ovulated ova was 2.00${\pm}$0.00, 2.86${\pm}$0.69 and 3.00${\pm}$1.22, respectively, after receiving 10, 15 and 20 mg ACAPE. The fertilized and cultured ova were able to develop into embryos up to the 32 cell stage. The present experiments demonstrated that multiple ovulations can be induced by CAPE and ACAPE successfully, and the ova resulted from the treatment retained the capability for further fertilization and embryonic development. These data provide new information to support the establishment of an in vitro culture system for future avian transgenesis studies.

• 한국가금학회지
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• 제28권2호
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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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• 제36권2_spc호
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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.

• 한국가금학회지
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• 제32권4호
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• pp.225-229
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• 2005

현재 가장 활발하게 진행되고 있는 형질전환 자 생산 연구방법은 배반엽단계 수정란에 농축한 virus를 주입하여 모자이크 형태의 $G_0$ 형질 전환체를 생산하고 이들을 이용하여 $G_1$ 형질전환 후대를 생산하는 방법이 가장 보편적으로 이용되고 있다. 상기의 연구방법은 완전한 형질 전환체를 획득하기 위해서는 수천수의 $G_1$을 생산하고 각각 유전분석을 수행하는 문제점을 가지고 있다. 이러한 문제점을 개선하고자 다음의 연구를 계획하고 수행하였다. 20nL의 농축된 GFP retrovirus를 1세포기 수정란에 주입하고, 주입한 유전자의 발현율과 수정란의 생존율을 배양 4일차 수정란에서 GFP의 발현과 배자의 생존 여부로 판정하였다. 연구결과는 배양 4일차 수정란의 생존율은 기존의 naked DNA 미세주입방법에 비해 다소 낮은 것으로 나타났으나 유의성은 없었다. 1세포기 수정란은 배반엽 단계 수정란과 달리 주입한virus의 유전자를 발현하지 않는 것으로 관찰되었다. 연구결과는 배반엽단계 수정란에 virus 미세주입 방법이 형질전환 닭 생산에 가장 효율적인 방법임 보여주고 있다.

• 한국가금학회지
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• 제31권4호
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• pp.293-298
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• 2004

본 연구는 기존의 형질전환 닭 생산방법 중의 하나인 1세 포기 수정란에 유전자를 직접 주입하는 유전자 미세주입방법을 개선할 목적으로 리포좀과 외래 표지 발현유전자인 GFP를 사용하여 외래유전자의 핵전이 효율성과 주입된 외래 유전자의 발현의 지속성을 닭의 배자에서 검증하고자 시도하였다 외래유전자는 배반엽 단계 혹은 1세포기 수정란의 세포질에 리포좀과 유전자의 혼합물 혹은 오직 유전자만을 미세주입을 하였다. 연구 결과들은 리포좀을 사용한 경우 naked DNA에 비하여 배반엽 단계와 1세포기 수정란 모두에서 효율적으로 외래 유전자를 핵내로 도입할 수 있음을 배양 3과 4일차 닭의 배자에서 GFP발현 양상을 통하여 확인하였다. 또한 주입된 외래 유전자에 의해 만들어진 GFP는 배자에서 일주일 정도 지속적으로 발현됨이 관찰되었다. 리포좀 방법은naked유전자 주입 방법에 비해 1세포기와 배반엽 단계 수정란 모두에서 효율적으로 외래 유전자를 핵내로 이동시키는 능력을 가지나, 주입된 유전자의 염색체 삽입에는 큰 영향을 미치지 않는 것으로 판단된다. 따라서 닭의 수정란에서 리포좀 방법은 외래유전자 도입에 유용한 수단으로 이용되어질 수 있을 것으로 사료된다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.20-21
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• 2003

Insulin-like growth factors (IGFs) are mitogenic peptide hormones that regulate embryonic development, postnatal growth and cellular differentiation in vertebrates IGFs are initially translated as pre-pro-peptides and then proteolytically processed to yield the mature IGFs and E-peptides. Like the C-peptide of pro-insulin, the E-peptides of pro-IGFs are generally believed to possess little or no biological activity other than their potential roles in the biosynthesis of the mature IGFs. Like human IGF-1, previous studies in our laboratory showed that the recombinant trout Ea4-peptide of pro-IGF-1 exhibited a dose-dependent mitegenic activity in cultured BALB/3T3 fibroblasts and other non-oncogenic transformed cells (Tian et al., 1999) We have also shown by in vitro and in vivo studies that Ea4-peptide possessed novel anti-tumor activities (Chen et al., 2002, Kuo and Chen, 2002; Kuo and Chen 2003). Recent results of studies conducted in chorionicallantoic membrane of developing chicken embryos revealed that Ea4-peptide of trout pro-IGF-1 also possesses a dose-dependent antiangiogenic activity. Together these results raised the question whether Ea4-peptide of trout pro-IGF-1 may affect heart and blood vessel development and hematopoiesis in fish embryos. (중략)