Transgenesis is a very powerful tool not only to help understanding the basics of life science but also to improve the efficiency of animal production. Since the first transgenic mouse was born in 1980, rapid development and wide application of this technique have been made in laboratory animals as well as in domestic animals. Although pronuclear injection is the most widely used method and nuclear transfer using somatic cells broadens the choice of making transgenic domestic animals, the demand for precise manipulation of the genome leads to the utilization of gene targeting. To make this technique possible, a pluripotent embryonic cell line such as embryonic stem (ES) cell is required to carry genetic mutation to further generations. However, ES cell, well established in mice, is not available in domestic animals even though many attempt to establish the cell line. An alternate source of pluripotent cells is embryonic germ (EG) cells derived from primordial germ cells (PGCs). To make gene targeting feasible in this cell line, a better culture system would help to minimize the unnecessary loss of cells in vitro. In this review, general methods to produce transgenic domestic animals will be mentioned. Also, it will focus on germ cell engineering and methods to improve the establishment of pluripotent embryonic cell lines in domestic animals.
Kim, Young-Hee;Cho, Sung-Whan;Ha, Chang-Su;Kang, Boo-Hyon
Toxicological Research
/
v.17
no.4
/
pp.241-248
/
2001
Exposure to 2-Bromopropane has been known to cause degeneration of male germ cells. However the mechanism underlying this process is poorly understood. The objective of this study was to determine whether or not the exposure of male Sprague-Dawley rats to 2-BP induces apoptosis in male germ cells. Male rats(N=3 or 4 in each group) were orally administered either with the corn oil vehicle (10 ml/kg body weight) or with 2-BP (3,500 mg/kg) once a day for 3 days. The presence of apoptosis was determined by TUNEL detection in situ and by an increase in DNA fragmentation. A low spontaneous incidence of apoptosis was observed in vehicle control animals, especially in pre-meiotic germ cells of stages I-VI and stages XII-XIV the seminiferous tubules. In 2-BP exposure rats, the incidence of apoptosis markedly increased at 4 h, reached a peak at 8 h (about 7-fold over control), and then decreased rapidly to control level by 48 h after the last administration. Although apoptosis induced by 2-BP occurred in all stages of germ cells, it was most pronounced in spermatogonia and early spermatocytes in stages I-VI and stages XII-XIV. Taken together, our results suggest that apoptosis is involved in the toxicity of testicular germ cells resulting in oligospermia or azoospermia after exposure to 2-BP.
This study was carried out to establish the in vitro short-term culture system of developing male germ cells by purifing germ cells of various stages. The decapulated testicular cells were incubated with collagenase (lmg/ml) and try psin (2.5mg/ml) in HBSS. After separating male germ cell, the separated germ cells were stained with heamatoxylin/eosin and determined developing stages under light microscopy. The purity of pachtene spermatocytes a and round spermatid were 85%, respectively. Yield of total male germ cells was highly variable between individuals, with a mean value of 3.5 to 4.5 ${\times}$ 10$^7$ cells/testis. Viability of the cell was over 97% after separation. In DMEM medium, the optimal cell number for culture is approximately 1 x 10$^5$ cells/dish, but low cell den-sities than 1 ${\times}$ 10$^5$ cell/dish showed a decreased cell viability. Furthermore, about :36.8% of pac-hytene cells was successfully cultured for 6 days and some of cells were developed to secondary spermatids and round spermatids. Therefore, our data suggested that this culture conditions will be utilize as a feasible tools to produce tran-sgenic livestock using techniques such as intrac-ytoplasmic injection and cell fusion.
The purpose of this study was to examine the effects of oral-administered sex hormone for hybrid sturgeon, bester juvenile. The bester juveniles (2 months after hatching) were received a diet containing various doses of $17\alpha$-methyltestosterone (MT) or estradiol-$17\beta$ ($E_2$) for 6 months. Somatic growth of bester sturgeon juvenile did not show significant differences between experimental and control groups (27.9-30.5 cm; 125.1-161.7 g), although survival percentages showed a decreasing tendency in MT-treated animals. By histological examination, germ cells were recorded as smooth type in MT-treated fish and uneven type of germinal epithelium in $E_2$-treated animals. Their sex ratios were 5:4:1 (male: female: undifferentiation) in control and low dose of MT-treated fish (1 mg/kg), and 9:1:0 in fish treated with high dose of MT (10 mg/kg), whereas the ratios were reversed by both low and high doses of $E_2$ treatment, recorded as 2:8:0. Gonadal areas were not significantly differed in all trials (424,600.4 - 1,039,656.3 ${\mu}m^2$). Total number of germ cells, number of germ cells per gonadal areas and number of germ cells per area were significantly higher to 144.7-148.7 cells/section, 374.0-408.5 $cells/mm^2$ and 1,599.5-1,670.9 $cells/mm^2$ in $E_2$ treatment than those of others (30.4-63.9 cells/section, 148.4-226.9 $cells/mm^2$ and 850.0-1,050.6 $cells/mm^2$), respectively. And somatic growth according to their gender was not significantly differed between male and female.
Male germ cell apoptosis has been extensively explored in rodent. In contrast, very little is known about their susceptibility to apoptosis stimuli of developing germ cell stages at the time when germ cell depletion after busulfan treatment occurs. Furthermore, it is still unanswered how spermatogonial stem cells are resistant to busulfan treatment. We examined the change of gene expression in detail using cDNA microarray analysis of mouse testis treated with busulfan. A subtoxic dose of busulfan (40mg/kg of body weight) transiently increased 228 mRNA levels among of the 8000 genes analyzed. TagMan analysis confirmed that the mRNA levels such as defensive protein, support protein, enzymatic protein, transport protein, and hormonal protein were rapidly increased. These results were re-confirmed by real-time PCR analysis. However, the expression levels of these genes induced by busulfan treatment were significantly reduced in control testis, indicating that both of male germ cells and somatic cells after busulfan treatment induces self-defense mechanism for protection of testicular cell death. Among them, we conclude that defense proteins play a key role in testis injury induced by busulfan.
To investigate the role of miR-1297 and the tumor suppressor gene PTEN in cell proliferation of testicular germ cell tumors (TGCT). MTT assays were used to test the effect of miR-1297 on proliferation of the NCCIT testicular germ cell tumor cell line. In NCCIT cells, the expression of PTEN was assessed by Western blotting further. In order to confirm target association between miR-1297 and 3'-UTR of PTEN, a luciferase reporter activity assay was employed. Moreover, roles of PTEN in proliferation of NCCIT cells were evaluated by transfection of PTEN siRNA. Proliferation of NCCIT cells was promoted by miR-1297 in a concentration-dependent manner. In addition, miR-1297 could bind to the 3'-UTR of PTEN based on luciferase reporter activity assay, and reduced expression of PTEN at protein level was found. Proliferation of NCCIT cells was significantly enhanced after knockdown of PTEN by siRNA. miR-1297 as a potential oncogene could induce cell proliferation by targeting PTEN in NCCIT cells.
A novel sterategy has been established to determine the origin of the Primordial Germ Cells (PGCs) in avian embryos directly and the developmental fate of the PGCs for the application to Poultry biotechnology. Cells were removed from 1) the centre of area pellucida, 2) the outer of area pellucida and 3) the area opaca of the stage X blastoderm (Eyal-Giladi & Kochav, 1976). When the cells were removed from the centre of area pellucida, the mean number of circulating PGCs in blood was significantly decreased in the embryo at stage 15 (Hamburger & Hamilton, 1951) as compared to intact embryos. When the cells were replenished with donor cells, no reduction in the PGCs number was observed. The removal of cells at the outer of area pellucida or at the area opaca had no effect on the number of PGCs. In case, another set of the manipulated embryos were cultured ex vivo to the hatching and reared to the sexual maturity, the absence of germ cells and degeneration of seminiferous tubules was observed in resulting chickens derived from the blastoderm in which the cells were removed from the centre of the area pellucida. It was concluded that the avian Primordial Germ cells are originated at the center of area pellucida. Developmental ability of the cells to differentiate into somatic cells and germ cells in chimeras were analyzed. Somatic chimerism was detected as black feather attributed from donor cells. Molecular identification by use of female - specific DNA was performed. It was confirmed that the donor cells could be differentiated into chimeric body and erythrocytes. Donor cells retained the ability to differentiate into germline in chimeric gonads. More than 70% of the generated chimeras transmitted donor derived gametes to their offspring indicating that the cells at the center of area pellucida had the high ability to differentiate into germ cells. A molecular technique to identify germline chimerism has been developed by use of gene scan analysis. Strain specific DNA fragments were amplified by the method. It would be greatly contributed for the detection of germline chimerism. Mixed- sex chimeras which contained both male and female cells were produced to investigate the developmental fate of male and female cells in ovary and testes. The sex combinations of donor and recipient of the resulting chimeras were following 4 pairs; (1) chimeras (ZZ/ZZ) produced by a male donor (ZZ) and a male recipient (ZZ), (2) chimeras (ZW/ZW) produced by a female donor (ZW) and a female recipient (ZW), (3) chimeras (ZZ/ZW) Produce by a male donor (ZZ) and a female recipient (ZW), (4) chimeras (ZW/ZZ) produced by a female donor (ZW) and a male recipient (ZZ). It was found that genetically male avian germ cells could differentiate into functional ova and that genetically female germ cells can differentiate into functional spermatozoa in the gonad of the mixed- sex chimeras. An ability for introduction of exogenous DNA into the PGCs from stage X blastoderms were analyzed. Two reporter genes, SV-$\beta$gal and RSV-GFP, were introduced into the PGCs. Expression of bacterial/gal was improved by complexing DNA with liposome detectedcc in 75% of embryos at 3 days embryos. At the embryos incubated for 1 day, expression of the GFP was observed all the embryos. At day 3 of incubation, GFP was detected in about 70% of the manipulated embryos. In case of GFP, expression of the transgene was detected in 30 %e of the manipulated embryos. These results suggested that the cells is one of the most promising vectors for transgenesis. The established strategy should be very powerfull for application to poultry biotechnology.
The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.
Shim, Sang Woo;Han, Dong Wook;Yang, Ji Hoon;Lee, Bo Yeon;Kim, Seung Bo;Shim, Hosup;Lee, Hoon Taek
Molecules and Cells
/
v.25
no.3
/
pp.358-367
/
2008
The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.
Proceedings of the Korea Society of Poultry Science Conference
/
2001.11a
/
pp.74-76
/
2001
Comparing to mammals, male bird has the homozygote ZZ and female has the heterozygote n. Therefore, the sex of fertilized eggs is defined by female chromosome constitution. Although this cytological observation had been established, the molecular and cellular mechanism of germ cell differentiation are essentially unknown in aves. Especially, the differentiation of germ cells in mixed-sex chimeras has not yet been clearly elucidated. Primordial germ cells, which are the progenitors of sperm or egg after sexual maturity, firstly arise in the epiblast and migrate to embryonic gonads through the blood vessel. During the embryo development, these PGCs differentiate in the pathway of mate or female, respectively and develop the sperm or egg cells after sexual maturity. In this paper, we confirmed that the female PGCs could migrate into the recipient male gonads after transferring and differentiate into germ cells in the embryonic stages. The primordial germ cells were isolated from the female embryonic gonads of 5.5-day-old incubation and re-injected into the male recipient embryos of 2-day-old incubation, which produced mixed-sex chimera in the germline. The finding in this study demonstrated the ability of migration and differentiation of gonadal primordial germ cells in mixed-sex chicken.
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.