Targeting lentiviral vectors to primordial germ cells(PGCs):An efficient strategy for generating transgenic chickens

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

Culture of Embryonic Stem Cell by Primordial Germ Cells of Down Producing Goat

[Objective] The paper was to establish embryonic stem cell system of goats. [Method] Numerous primordial germ cell colonies were derived from gonadal ridge and the surrounding tissues in 20 millimeter fetuses of down producing goat. Primordial germ cells and goats embryonic fibroblasts obtained from conceptus of equivaient gestational age were co-cultured. [Result] The colonies showed some characteristics of embryonic stem cells, such as the morphology of nest-like, they continued to be AKP positive and the ability to be continuously passed [Conclusion] These cells were pluripotent and ES-like cells.

Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

Background:NANOG is a core transcription factor(TF)in embryonic stem cells(ESCs)and primordial germ cells(PGCs).Regulation of the NANOG gene by TFs,epigenetic factors,and autoregulatory factors is well characterized in ESCs,and transcriptional regulation of NANOG is well established in these cells.Although NANOG plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied.Therefore,we investigated the mechanism that regulates transcription of the chicken NANOG(cNANOG)gene in PGCs and ESCs.Results:We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis.Then,we measured the promoter activity of various 5′flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay.cNANOG expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in PGCs.The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ESCs.Furthermore,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of cNANOG.Conclusions:We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner.This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

Developmental potency of mouse primitive ectoderm cells, embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c.male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c.blastocysts.Sixteen (61.5) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells;four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts.However,no overt chimaera was obtained with either 5.25-day or 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells.GPI analysis of mid-gestation conceptuses developed from injected blastocysts showedthat 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus.Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells,and their developmental potency becomes limited within a short period of time in normal development.

α-ketoglutarate promotes the specialization of primordial germ cell-like cells through regulating epigenetic reprogramming

There is growing evidence that cellular metabolism can directly participate in epigenetic dynamics and consequently modulate gene expression.However,the role of metabolites in activating the key gene regulatory network for specialization of germ cell lineage remains largely unknown.Here,we identified some cellular metabolites with significant changes by untargeted metabolomics between mouse epiblast-like cells(EpiLCs)and primordial germ cell-like cells(PGCLCs).More importantly,we found that inhibition of glutaminolysis by bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide(BPTES)impeded PGCLC specialization,but the impediment could be rescued by addition ofα-ketoglutarate(αKG),the intermediate metabolite of oxidative phosphorylation and glutaminolysis.Moreover,adding aKG alone to the PGCLC medium accelerated the PGCLC specialization through promoting H3 K27 me3 demethylation.Thus,our study reveals the importance of metabolite aKG in the germ cell fate determination and highlights the essential role of cellular metabolism in shaping the cell identities through epigenetic events.

Chicken Mesenchymal Stem Cells as Feeder Cells Facilitate the Cultivation of Primordial Germ Cells from Circulating Blood and Gonadal Ridge

Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.

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.

Effects of 17beta-estradiol on cell migration in male chicks distribution of primordial germ

Aim： To assess whether exogenous estradiol has any effect on migration of primordial germ cells （PGCs） in the chick. Methods： Fertilized eggs were treated with 17beta-estradiol （E2） （80 lag/egg） at stage X （day 0 of incubation）, stages 8-10 （incubation 30 h） and 13-15 （incubation 55 h）. Controls received vehicle （emulsion） only. Changes in PGC number were measured on different days according to developmental stages. Results： In male right gonads, but not in female left gonads, at stages 28-30 （incubation 132 h） significant decreases in the mean number of PGCs aggregating were observed compared with the controls （P 〈 0.05） while the total PGC number in the right and left gonads at each stage did not change （P 〉 0.05）. Conclusion： The present study provides evidence that E2 has significant effects on the localization of PGCs in male right, but not female left, gonads of chicken embryos at stages 28-30, compared with controls. （Asian J Andro12008 Mar; 10： 243-248）

Reprogramming of germ cells into pluripotency

Primordial germ cells(PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

Improved Isolation and Culture of Embryonic Germ Cells from Guanzhong Dairy Goat

A total of 219 embryonic-germ-cell-like （EG-like） clumps were derived from 15 selected goat fetuses. Isolation of primordial germ cells （PGCs） based on co-culture with primary goat embryonic fibroblast showed no difference from traditional feeder layer-based culture method used in mouse and human. The putative primary EG colonies were multilayer clumps of compact cells with unclear cell-cell boundaries. Three subculture methods of goat EG-like colony, traditional enzymatic digestion, mechanical cutting and combination of the both, were compared in this study. As a result, EG-like colonies traditionally disassociated with collagenase 1V could be subcultured for up to 4 passages. And the mechanically disaggregated EG-like colonies were successfully maintained 9-12 passages with or without enzymatic treatment. The pluripotency of the EG-like colonies was identified by their specific marker staining, spontaneous differentiation and embryoid bodies （EBs） formation in vitro. Most goat EG-like colonies （〉 80%） were AKP positive and immunocytochemically characterized with positive SSEA-1, Oct-4 and c-kit staining but SSEA-4. Under the condition of delaying passage, goat EG-like cells could differentiate into fibroblast-like, epithelium-like, and neuron-like cells. In addition, EBs could be obtained successfully in routine hanging drop culture. The serum free culture system （feeder layer-based） used in this study was suitable for keeping PGCs and EG-like cells in their undifferentiated condition, but failed to converse them to immortal cells. These results indicated that mechanical cutting is an effective method for passaging goat EG cell colonies. However, the microenvironment of conversing EG cells to immortal cells is still unclear.

山羊PGCs用于分离与克隆类ES细胞

选择健康成年本地白山羊 ,自然发情、配种后 4 4 d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 ,采用传统方法和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个类 ES细胞集落 。

细胞因子对鸡胚胎原始生殖细胞(EPGCs)增殖的影响

采用MTT法分别检测mLIF、bFGF、hSCF、hIL-11四种细胞因子协同作用对体外培养的第19、28期鸡EPGCs生长的影响。结果表明与对照组相比较,19期的EPGCs体外培养72h后,mLIF、hSCF、bFGF、hIL-11对鸡EPGCs的增殖影响显著（P〈0.05）。mLIF的最佳作用剂量是10-20ng/ml,hSCF的最佳作用剂量是15-20ng/ml,bFGF的最佳作用剂量是10-20ng/ml。hSCF、bFGF的联合使用优于单因子作用的结果（P〈0.05）。单独使用hIL-11时,细胞的增殖情况比其他三因子单独使用的效果较差,但OD均值有随剂量增高而上升的趋势。在与其他因子联合使用的情况下,hIL-11的最佳作用剂量为0.10-0.20ng/ml。

饲养层及细胞因子对鸡PGCs体外培养的影响

原始生殖细胞(PGCs)具备发育全能性,而且数量较多,与数量较少的内细胞团(ICM)相比更有利于进行分离克隆。不同动物的PGCs的生长条件不同,因此建立适宜的培养体系是PGCs培养的关键。对饲养层细胞及细胞因子对鸡PGCs体外培养的影响作了简单的介绍。

鸡原生殖细胞(PGCs)的提纯及其超低温保存

利用密度梯度离心从孵化51～56h的鸡胚血液中提纯PGCs，通过形态鉴别和过碘酸－希夫试剂染色方法判定提纯的PGCs的纯度在80％左右。将提取到的PGCs放入到含有10％二甲基亚砜的TCM－199中，在液氮中进行保存。将超低温冷冻保存了1个月、6个月和12个月的PGCs（各3管）分别复苏后，用台盼蓝染色，通过红细胞计数板计数鉴定冷冻保存了不同时间的PGCs的平均存活率分别为91．7％、92．6％和91．9％。结果表明所采用的冷冻保存的方法是可行的。所采用的超低温冷冻保存方法简单易行，为今后以PGCs为目的细胞进行超低温冷冻，而后制作种系嵌合体进行保种的研究提供有利的实践基础。

内蒙古绒山羊原始生殖细胞(PGCs)的培养与鉴定

本试验从内蒙古白绒山羊胎儿的生殖嵴中分离出原始生殖细胞体外培养并进行了鉴定。为建立内蒙古绒山羊原始生殖细胞(pri mordial germcells,PGCs)的培养体系,试验对比了A、B、C3组不同的培养条件对内蒙古绒山羊原始生殖细胞传代数的影响。RT-PCR鉴定结果表明,β-actin、hTERT、GAPDH、Nanog、Oct3/4为阳性、AKP染色为阳性;免疫荧光检测胚胎干细胞特异标志物Oct3/4、SSEA-3、SSEA-4呈阳性。未添加任何因子的A培养体系仅能传1代,添加LIF及Insulin的B培养体系可以传至4代,添加LIF、Insulin及优质胎牛血清的C组培养体系可以传至9代。

哺乳动物PGCs用于胚胎干细胞培养的研究

本文综述了哺乳动物原始生殖细胞 (PGCs)的研究进展 ;归纳了影响用 PGCs分离胚胎干细胞培养的因素 ;阐述了在 PGCs用于胚胎干细胞培养研究中存在的问题及应用前景。

猪原始生殖嵴细胞(PGCs)建系因素的研究

从五指山猪(WSZP)近交系第8～13代培育群中,先后选用21头5～10月龄青年母猪,分别于授精后25～30d采集胎儿106个,进行原始生殖嵴(PGCs)细胞分离、培养等建系技术研究。以DMEM+F10(1∶1)为基础培养液,按添加或不添加生长因子,将培养液分为A、B、C3种,并以STO细胞作饲养层,在38℃、5.0%CO2和湿润的气相中进行培养建系。结果获得胚胎生殖嵴细胞(EG)细胞系6个细胞株,其中1个EG细胞株传至11代、2个传至5代、1个传至4代、2个传至3代冻存。并进行了AKP染色、体外分化、冷冻-解冻复苏和嵌合体制作等鉴定研究。研究发现:不同胚龄对EG细胞建系具有一定影响,不同培养液对EG细胞建系效果不同,STO细胞饲养层的质量是建株、传代、冷冻-解冻复苏的关键因素之一。EG细胞系的初步建立,为今后筛选进入种系的EG细胞系、实施体外基因操作提供了可能。

建立鸡EPGCs单细胞克隆株初探

目的 探索鸡EPGCs单细胞克隆建立细胞系的可能性,并对其生物学特性进行鉴定.方法 采取19期和28期的鸡EPGCs,体外培养传至第4代的细胞聚合体,用胰酶消化将细胞分散成单细胞悬液,将单个细胞接种至96孔板,每个孔内接种1个细胞,生长出的细胞集落用胶原酶消化传代,细胞化学法和免疫荧光法检测多向分化细胞的表面标志物;常规染色体核型检查.结果 接种的288个单细胞中有9个扩增,其中7个传至第2代,2个传至第4代,克隆形成率为3.1%.扩增出的2～4代单细胞克隆能稳定增殖不分化,具有正常二倍性染色体核型、碱性磷酸酶阳性、阶段特异性胚胎抗原（SSEA）-1等特征性细胞表面标志物呈阳性;离体情况下具有形成类胚体和类上皮样细胞的能力.结论 建立鸡EPGCs单细胞克隆细胞系是可行的,其生物学特性稳定.

鸡WDR5基因真核表达载体构建及对PGCs形成相关基因表达的影响

【目的】对鸡色氨酸天冬氨酸重复域5(WD repeat domain 5,WDR5)进行生物信息学分析,构建真核表达载体,并检测其对原始生殖细胞(Primordial germ cells,PGCs)形成相关基因表达的影响,探索WDR5在鸡PGCs形成中的调控作用。【方法】利用ProtParam、NCBI保守结构域分析、Uniport、TMHMM Server v.2.0和SignalP在线网站,分析WDR5的氨基酸序列、保守结构域、亲疏水性、跨膜结构域和信号肽表达。利用SWISS-MODEL进行同源建模预测其三级结构,借助String网站进行互作蛋白的预测。构建重组载体pcDNA3.1-WDR5-GST,进行双酶切和测序鉴定;将重组载体pcDNA3.1-WDR5-GST转染PGCs,采用实时荧光定量PCR(RT-qPCR)和Western blot检验WDR5重组蛋白在PGC中的表达情况;用RT-qPCR检测过表达WDR5对PGCs形成相关基因(DDX4、C-KIT、BLIMP1、LIN28B和PRDM14)表达水平的影响。【结果】WDR5蛋白含有1个高度保守的WD40结构域,内含7个典型的WD40重复序列;其亲水性高、不含跨膜结构域、无信号肽表达,可用于构建重组真核表达载体。同源建模和互作蛋白预测显示WDR5高度保守,互作蛋白为类ASH2蛋白(ASH2 Like,ASH2L)、RB结合蛋白5(RB Binding Protein 5,RBBP5)、DPY30和KAT8调控因子NSL复合物亚基3(KAT8 Regulatory NSL Complex Subunit 3,KANSL3)。双酶切和测序结果表明,重组载体pcDNA3.1-WDR5-GST构建成功。RT-qPCR结果显示PGC中重组载体可使WDR5过量表达;Western blot结果表明,PGC中可表达重组蛋白。WDR5过表达可使PGCs形成相关基因的表达水平升高。【结论】构建的WDR5重组载体在鸡PGCs中能够表达;WDR5基因表达上调后,PGCs形成相关基因表达水平升高,推测其可调控鸡PGCs的形成。

猪原始生殖嵴(PGCs)细胞分离、建系培养

Inbred of Wu Zhishan miniature pig(WZSP)at 5 to 8 Mouths of age,which were used as embryo donors.The total 75 fetuses was collected from 14 donors at day 25 to 29 of pregnancy(estrus=day 0),which was used for in vitro culture,clone,isolation,passage of porcine embryonic germ(PEG).The results were shown that:(1)The PGCs of fetuses between days 2729 of pregnancy(estrus=0)was easily to collect and more suitable to set up PEGs.(2)The PEGs clones with WZSP was coming out 2 days early than Shim(97),Piedrahita(98)and Mueller(99)reported previously.(3)PEG′s will be survival in vitro after thawing.