Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.     

Perspectives on avian stem cells for poultry breeding

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.     

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.     

Primordial germ cell-mediated transgenesis and genome editing in birds

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.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens.     

Attempt to produce nuclear transferred primordial germ cells using electrofusion in domestic chicken

As a step to develop a somatic nuclear transfer technique for avian species, an attempt to produce somatic nuclear transferred primordial germ cells (PGC) in the domestic chicken was carried out. Primordial germ cells and embryonic blood cells (EBC) were collected from 2‐day‐old embryos and the nuclei were transferred from EBC into PGC by electrofusion. The most efficient pearl chain was developed when a 350‐V/cm AC field was applied for 60 s. Cell fusion between PGC and EBC was most effective when 4‐kV/cm DC pulses, 60 µs pulse width, were applied three times to a cell suspension dispersed in 0.2 or 0.25 mol/L saccharose solution. The present results provide basic information for the production of somatic cell nuclear transferred chickens using PGC as the nuclear recipient.     

Donor primordial germ cell-derived offspring from recipient germline chimaeric chickens: Absence of long term immune rejection and effects on sex ratios

1. Germline chimaeric chickens were produced by the transfer of primordial germ cells, and the generation of donor-derived offspring was examined for a maximum of 146 weeks. 2. The frequencies of donor-derived offspring from the chimaeras were 47% to 97%, and no apparent changes in frequency were observed with increasing age during the test period. 3. Differentiation of donor primordial germ cells into functional gametes appeared to be restricted to a degree at some developmental stage in the gonads of chimaeric chickens of the opposite sex.     

Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos

Background

Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem (ES) cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES cells into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator (PMM), and trace the fate of the injected ES cells.

Results

We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly.

Conclusions

These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.     

The Influencing Factor of In Vitro Fertilization and Embryonic Transfer in the Domestic Fowl (Gallus domesticus)

This study was conducted to explore the influencing factors of ova in vitro fertilization (IVF) and transfer of the fertilized ova into the oviduct of recipient hens. The efficiency of fertilization was compared using three aspects: (i) the different time of ova collection and transfer, (ii) egg‐laying period of recipient hen; and (iii) semen volume. The following results are observed: 72%, 40% and 0% of ova were found in ovarian sac in 30～40 min, 50～60 min and more than 90 min post‐oviposition, respectively; 20%, 18%, 14% and 5.8% of ova were fertilized with 0.1, 0.2, 0.5 and 1.0 ml semen, respectively; and 33% and 100% of healthy chickens were hatched from fertile ova with 0.1 and 0.5 ml of semen, respectively. All oocytes obtained from ovary and mid‐oviduct were unfertilized. Embryos were transferred into recipient hens 30 min ± 10 min post‐oviposition, and 70% of shelled eggs were produced. There were no eggs produced in the other transfer times. This demonstrated that live chicken can be obtained by IVF of ova collected shortly after oviposition. It was important that the ovum was transferred into the oviduct infundibulum of recipient hens immediately or shortly after oviposition.     

鸡胚胎生殖嵴中原始生殖细胞的分离培养研究

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     

CRISPR/Cas9 knock-in介导的输卵管特异性表达转基因鸡研究进展

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。     

胚胎干细胞及种系嵌合体的研究进展

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     

胚胎干细胞向生殖细胞分化的研究进展

生殖细胞来源于原始生殖细胞,其在体内的分化机制已基本清楚。随着生殖细胞研究的不断深入,通过体外诱导途径获得生殖细胞已成为现实。将胚胎干细胞体外分化为上胚层样细胞,进而分化为原始生殖样或原始生殖细胞。将它们迁移入胎儿生殖腺,具有减数分裂及产生精子能力,与卵子结合移入缺生精管的生殖腺的新生鼠可以产生健康后代。为此,体外诱导生殖细胞的成功,进一步阐明了胚胎干细胞向生殖细胞分化的机制,为更有效利用干细胞造福人类奠定了基础。     

ZB transposon and chicken vasa homologue (Cvh) promoter interact to increase transfection efficiency of primordial germ cells in vivo

ABSTRACT

1. 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.     

Manipulation of fish germ cell: visualization, cryopreservation and transplantation

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.     

Generation of Buffalo (Bubalus bubalis) Transgenic Chimeric and Nuclear Transfer Embryos Using Embryonic Germ-Like Cells Expressing Enhanced Green Fluorescent Protein

The possibility of producing transgenic buffalo embryos by chimera and nuclear transfer (NT) using buffalo embryonic germ (EG)‐like cells expressing enhanced green fluorescent protein (EGFP) has been explored in this study. Buffalo EG‐like cells and fibroblasts with two to eight passages were transfected with the lined plasmid (pCE‐EGFP‐IRES‐Neo‐dNdB) using Lipofectamine^TM 2000 and selected by culturing in 200 μg/ml G418 for 6–8 days. G418 resistant fibroblasts and EG‐like cells were used for embryo chimera and NT. To produce blastocysts by chimera, 8–16 cells embryos were injected with EG‐like and fibroblast cells. Then, to produce blastocysts by NT, in vitro maturated oocytes were enucleated and afterwards EG‐like/fibroblast cells transferred into the perivitelline space. No statistical differences were observed for the total blastocyst produced by the chimeric method, using EG‐like and fibroblasts as donor cells, resulting on an accomplishment of 35.6% vs 33.3%, respectively. Nevertheless, besides from the 37 blastocysts produced, 23 (62.2%) from EG‐like cells expressed EGFP, none of blastocysts from foetal fibroblasts expressed this protein. When the NT method was used, no statistical difference among different generations was observed in the percentage of oocytes fused, cleaved, and developed to blastocysts after NT for EG‐like cells. On average, the percentage of oocytes fused, cleaved, and developed to blastocysts after NT was respectively 81.8%, 67.7% and 10.7%. For the expression of EGFP, from the 12 blastocysts produced by NT, 7 of them were positive, while none of NT embryos from EGFP positive fibroblasts developed to blastocysts. Results of the present study clearly demonstrated that gene transfected buffalo EG‐like cells have the ability to form chimeric embryos after injecting into buffalo early embryos and reprogramming ability after NT, which can be employed to produce transgenic buffalos through either embryo chimera or NT.     

禽类多能性干细胞研究进展

胚胎干细胞是未分化的具有增殖和自我更新能力的细胞,并且能分化成所有类型的体细胞以及生殖细胞。它们提供了早期胚胎分化的体外模型,也是基因操作的重要靶细胞。禽类多能性干细胞培养最重要的应用领域是以干细胞体外遗传修饰、鉴定为技术平台的家禽转基因技术。通过此技术对禽类基因进行遗传修饰与操作,在胚胎发育基础研究、转基因禽类生产及家禽育种等方面有巨大的应用前景。但是禽类多能性干细胞培养的许多基本问题仍亟待解决,如探索其建系的培养条件、揭示其维持多能性和增殖能力的分子机制等。文章综述了禽类多能性干细胞的分离方法、体外分化能力、嵌合体形成以及基因修饰方面的研究进展及目前的研究局限。