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1.
Effect of soft X-ray irradiation on the migratory ability of primordial germ cells in chickens 总被引：1，自引：0，他引：1
1. The present study was conducted to elucidate the effect of soft X-ray irradiation on the migratory ability of primordial germ cells (PGCs) to the germinal ridges of chicken embryos. 2. PGCs (Barred Plymouth Rock, BPR) were isolated from embryonic blood and irradiated with soft X-rays for 1-10 min. Then, the PGCs were transfected in vitro with GFP gene by lipofection. The manipulated PGCs were transferred to recipient embryos (White Leghorn, WL) and migration to the germinal ridges was analysed by examining GFP gene expression in the gonads of recipient embryos under UV light at x40 magnifications. The expression of GFP gene was detected in all the gonads of recipient embryos examined up to 10.5 d of culture. 3. Migration of PGCs irradiated with soft X-rays to the germinal ridges was also confirmed by detecting a single nucleotide polymorphism in the D-loop region of the mitochondrial DNA of BPR and WL chickens. Freshly collected PGCs (BPR) were transferred to the bloodstream of recipient embryos (WL). The fate of the transferred donor PGCs was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in BPR and WL used in this study. Transferred donor PGC-derived cells were detected in all the gonads of 17-d cultured embryos by PCR. 4. The results suggest that PGCs irradiated with soft X-rays still retain the ability to migrate to the germinal ridges of recipient embryos. 相似文献
2.
Chojnacka-Puchta L Kasperczyk K Płucienniczak G Sawicka D Bednarczyk M 《Polish journal of veterinary sciences》2012,15(1):181-188
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. 相似文献
3.
胚胎干细胞及种系嵌合体的研究进展 总被引：1，自引：0，他引：1
胚胎干细胞是着床前的囊胚内细胞团或早期胎儿的原始生殖细胞经体外分化抑制培养建立的多能性细胞系 ,具有与胚胎细胞相似的形态特征和分化潜能 ,体外培养时保持未分化状态 ,可以传代增殖。改变维持胚胎干细胞不分化的培养条件 ,胚胎干细胞可自发分化成多细胞结构。在一定诱导下 ,胚胎干细胞可向多个方向分化 ,并生成多种功能细胞。胚胎干细胞注入到胚泡期胚胎或与桑椹期胚胎聚合 ,可以参与包括性腺在内的各种组织的嵌合体的形成。胚胎干细胞在细胞分化与调控 ,胚胎发育 ,遗传病 ,肿瘤 ,免疫和组织或器官移植等研究中显示着广泛的应用前景。而种系嵌合体的获得是实现 ES细胞途径的决定步骤 ,低的种系嵌合率则是制约 ES细胞应用的关键。提高供体 PGCs在受体生殖腺中的比例 ,缩短 ES细胞的体外培养时间 ,以及注入早期发育阶段的受体胚胎等都能提高种系嵌合率。文章从多个方面综述了胚胎干细胞的最新研究成果 ,并着重以禽类 ES细胞为例论述了种系嵌合体的检测方法 ,种系嵌合率的影响因素以及提高种系嵌合率的方法 相似文献
4.
ABSTRACT1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter.2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2–3 or via blood vessel at HH stage 13–14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13–14 were significantly higher than that at HH stage 2–3.3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence.4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13–14 may be a more efficient route for PGCs transfection in vivo. 相似文献
5.
Yoshiaki NAKAMURA Mariko TASAI Kumiko TAKEDA Keijiro NIRASAWA Takahiro TAGAMI 《The Journal of reproduction and development》2013,59(6):580-587
The Japanese quail (Coturnix japonica) is a valuable bird as both
an experimental animal, for a wide range of scientific disciplines, and an
agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs
would be a feasible strategy for the conservation of both male and female fertility
cells in Japanese quail. However, the effects of freeze-thaw treatment on viability,
migration ability and germline transmission ability of quail PGCs still remain
unclear. In the present study, male and female PGCs were isolated from the blood of
2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196
C for 77–185 days, respectively. The average recovery rate of PGCs after
freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly
lower than that of the control group (P<0.05) (85.5% vs. 95.1%).
Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into
recipient embryos migrated toward and were incorporated into recipient gonads,
although the number of PGCs settled in the gonads was 48.5% lower in the frozen group
than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that
one female and two male recipients produced live progeny derived from the
frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than
that of unfrozen controls, but the difference was not significant (4.0
vs. 14.0%). These results revealed that freeze-thaw treatment
causes a decrease in viability, migration ability and germline transmission ability
of PGCs in quail. 相似文献
6.
Mitsuru NAITO 《Animal Science Journal》2003,74(3):157-168
The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary. 相似文献
7.
山羊PGCs用于分离与克隆类ES细胞 总被引：11，自引：1，他引：10
选择健康成年本地白山羊，自然发情，配种后44d取胎儿，以传统的原始生殖细胞（PGCs)分离与克隆的方法和PGCs与其胎儿生殖嵴周围组织细胞共同培养的方法获得类胚胎干细胞（类ES细胞），并对山羊类ES细胞在不同饲养层上进行培养。结果表明，采用传统方法与共培养的方法并添加细胞因子均能分离获得类ES细胞。分离获得的类ES细胞在同源（山羊）胎儿细胞饲养层上生长效果较好，可传4代或5代，而在小鼠原代成纤维细胞饲养层上类ES细胞仅传3代。另外，共培养不添加细胞因子组仅获1个ES细胞集落，传代后丢失。 相似文献
8.
9.
Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells(PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds,including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs.Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans. 相似文献
10.
Hiroshi Kagami 《Animal Science Journal》2016,87(9):1065-1075
Stem cells have prulipotency to differentiate into many types of cell lineages. Recent progress of avian biotechnology enabled us to analyze the developmental fate of the stem cells: embryonic stem cells / primordial germ cells (PGCs). The stem cells were identified in the central area of the area pellucida of the stage X blastoderms. These cells could be applied for production of germline chimeras and organ regeneration. Generation of medical substrate in transgenic chickens has considerable interests in pharmaceuticals. Sex alteration of the offspring should be enormously beneficial to the poultry industry. Fertilization of the sex‐reversed sperm could lead to sexual alteration of the offspring. These strategies using stem cells / PGCs should be one of the most powerful tools for future poultry breeding. 相似文献
11.
To compare the hepatic function of broiler and layer chickens (Gallus gallus domesticus) at various embryonic stages and hatching, the total lipid and triacylglycerol (TG) content were determined. The chicken embryos accumulated a large amount of lipids in the liver and the total lipid content gradually increased toward hatching, though no significant difference was observed between broilers and layers. The TG contents in the liver increased considerably with developmental stage. At embryonic day 14 the TG content in the liver was similar between broilers and layers; thereafter, it was 1.3 and 2.2 times higher in broilers than in layers at embryonic day 18 and for newly hatched chicks, respectively. Chick embryos accumulate an excessive amount of cholesterol ester in the liver, but cholesterol ester is replaced by TG after hatching. The results of hepatic TG contents in the present study suggest that the development of the hepatic function between broilers and layers may already differ at embryonic stages. 相似文献
12.
在鸡胚孵化的 19期以Ficoll密度梯度离心和酶解离两种方法分离生殖嵴中的原始生殖细胞 (PGCs)。探索在生殖嵴中PGCs分离、培养的适宜方法 ,以获得较多数量 ,较高活力的PGCs作介导生产转基因鸡。在倒置显微镜下进行形态观察 ,台盼兰染色比较存活时间 ,PAS特异染色法识别鉴定PGCs。结果表明两种分离方法均能分离到一定数量的PGCs细胞。与Ficoll密度梯度离心法相比 ,酶解离法分离到的PGCs的相对数量较多 ,存活时间较长 ,是一种较可行的分离方法。在鸡胚孵化的第 19期 ,PGCs大量聚集在肢体后端的生殖嵴原基处 ,此时的生殖嵴大小已达一定程度 ,分离其中的PGCs操作简便 ,有较强的可操作性 ;提取出的PGCs为转基因鸡的生产提供了介导材料 相似文献
13.
14.
A. Rahman M. Syduzzaman A. Khaliduzzaman S. Fujitani A. Kashimori T. Suzuki 《British poultry science》2020,61(2):209-216
ABSTRACT1. Sex-specific variations in early embryonic development rates may pre-empt later variations in embryonic development through to pipping and hatching. Given that erythropoiesis (blood production) can be equated with early embryonic growth rate, it was hypothesised that blood pigment haemoglobin can act as a specific spectral fingerprint for changes in growth rate. Moreover, by measuring longitudinal, rather than lateral, spectral transmission through the egg, a more consistent spectrum with a higher signal-to-noise ratio could be captured.2. Longitudinal visible transmission (T575/T598 ratio), which is sensitive to haemoglobin, was used to monitor sex-specific early embryonic development rate in white layer chicken eggs from d 0 to 8 of incubation. The sex of these eggs was subsequently confirmed two days after hatching.3. Embryonic development was detectable from d 3 (72 h) of incubation, 36 h earlier than previously reported lateral spectral measurements, supporting the greater sensitivity of longitudinal measurements.4. At d 3, the mean T575/T598 ratio for male embryos was significantly lower (P < 0.001) (i.e. higher absorbance of haemoglobin) than for female embryos, which was thought to be due to sex-differences in early embryogenesis. On the other hand, female embryos had a significantly lower (P < 0.05) mean T575/T598 ratio than male embryos at d 7 of incubation, presumably due to the combined effects of oestrogen synthesis receptors and enzymes on erythropoiesis in female embryos at this time.5. In conclusion, the proposed methodology has the sensitivity to differentiate sex-specific embryonic development rates during early incubation and the potentiality to advance precision incubation management and poultry research. 相似文献
15.
利用动物生物反应器生产重组蛋白是一种具有应用前景的生物技术。鸡输卵管生物反应器是理想的动物生物反应器之一,其优点在于表达的外源蛋白能够分泌到蛋清中,可避免蛋白提取过程中对鸡本身造成伤害,同时蛋清成分简单,便于后期的纯化。目前利用慢病毒结合原始生殖细胞(PGCs)制备转基因鸡被认为是最可行的方法,但因外源基因随机整合且生殖系传递效率较低,使转基因鸡研究受到技术上的限制。而2013年问世的CRISPR/Cas9基因敲入(CRISPR/Cas9 knock-in)技术能够使外源基因精准定向插入基因组特异性位点,这对生产输卵管特异性转基因鸡具有重大意义。文章综述了鸡输卵管反应器的研究进展、CRISPR/Cas9 knock-in技术在输卵管特异性表达转基因鸡研究和鸡育种领域的应用现状,并指出了目前存在的问题和相应的解决办法。 相似文献
16.
为了研究种蛋储存期对孵化性能的影响,试验以散养东北当地土鸡种蛋为研究对象,研究不同保存期(1～3 d、4～7 d、8～15 d和16～30 d)种蛋孵化性能的变化。结果表明:东北当地散养土鸡种蛋的最佳保存时间为7 d,种蛋保存时间在1～3 d和4～7 d之间的受精蛋孵化率变化不明显,保存超过7 d的种蛋受精蛋孵化率显著降低(P<0.05),且保存时间越长受精蛋孵化率越低;随着保存时间的延长,胚胎死亡率增高,保存1～15 d对胚胎早期死亡率影响不大,保存时间超过15 d会导致中后期死胚率增高。说明保存时间对种蛋孵化性能起关键性作用。 相似文献
17.
Production of quail-chick chimaeras by blastoderm cell transfer 总被引：2，自引：0，他引：2
1. Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos. 2. Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting. The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos. The chick embryos were then cultured in recipient eggshells. 3. Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells. The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively. 4. Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity. In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching. The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos. 5. This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens. 相似文献
18.
分别对取自50~95日龄水牛胎儿的原生殖细胞和前精原细胞进行体外培养,观察其生物学行为,并检测其碱性磷酸酶(AP)活性和Oct-4蛋白特性,探讨利用这些生殖细胞建立干细胞系的可行性和检测方法。结果表明水牛原生殖细胞及前精原细胞分别在体外培养时,均能形成细胞克隆;克隆与周围细胞分界明显,但克隆中细胞相互间界限不清;部分克隆有分隔现象,形如多个克隆共同组成一个大克隆;细胞克隆均至少能培养4代以上;原生殖细胞和前精原细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中部分克隆表现为AP假阳性。研究结果显示水牛原生殖细胞和前精原细胞均可用于建立干细胞系;体外培养时,AP活性和Oct-4蛋白不适宜用来检测这些细胞及其来源的细胞克隆。 相似文献
19.
The aim of this study was to explore the hepatic lipolysis in broiler chickens with different fat deposition during embryonic development. The mRNA expression of CPT-1 (carmitine palmtoyltransferase-1), PPARα (peroxisome proliferator-activated receptor alpha) and LPL (lipoprotein lipase) genes were determined using Real time RT-PCR. The start of incubation was called day 1 (E1) and after hatching called day 1 (H1). On incubation days 9 (E9), 14 (E14) and 19 (E19) as well as at hatching (H1), samples of liver were collected. Blood samples were obtained during days 14 (E14) and 19 (E19) of embryonic development and at hatching. This study showed that serum TG (triglycerol) decreased and TC (total cholesterol) and NEFA (non-estered fatty acid) increased during embryonic development. The expression of CPT-1, PPARα and LPL genes exhibited different developmental changes. For example, little LPL gene was expressed at hatching and PPARα gene expression peaked before hatching. However, CPT-1 gene exhibited no significance during the embryonic development. Our results showed that expression of these genes in Arbor Acres (AA) broilers was significantly higher than that in San Huang (SH) broilers. Therefore, this study suggested that hepatic lipolysis in broiler chickens exhibited developmental changes during embryogenesis and breed difference which may be one of the factors in the fat deposition difference between fat line and lean line broilers during embryonic development. 相似文献
20.
鸡早期胚胎精原细胞和睾丸发生发育关系的研究 总被引：6，自引：2，他引：6
采用连续切片技术，研究了15～45期(孵化50h～19d)鸡早期胚胎发育过程中精原细胞与睾丸发生发育的关系。结果显示：孵化第22～28期(第3．5～5天)，原始生殖腺内的原始生殖细胞(PGCs)胞质内的糖原颗粒开始分解；第29期(孵化第6天)，鸡胚性腺开始分化，PGCs的糖原颗粒进一步分解；第31期(孵化第7天)，PGCs内的糖原颗粒完全消失。在雄性，性腺开始显示睾丸特征，PGCs分化为精原细胞；第34期(孵化第8天)，睾丸内曲精细管索开始形成，呈实心状，精原细胞位于其中；第35～37期(孵化第9～11天)，曲精细管索数量逐渐增加，索内精原细胞体积较大，呈圆形单层排列，且已分化出支持细胞，但数量不多，形态不易分辨，间质内有少量间质细胞；第38～40期(孵化第12～14天)，可见到典型的曲精细管，精原细胞数量增加，支持细胞亦增多；曲精细管间的间质细胞成群分布。第40～45期(孵化第16～19天)，两侧睾丸大小略有差异，右侧稍大于左侧，精原细胞数量明显增多，呈葡萄串状分布在曲精细管的中央；曲精细管的管腔已经形成，精原细胞已分层排列。 相似文献