Four kinds of ENU-induced white spot mice and chromosome locations of the mutant genes

After the accomplishment of the Human Genome Project, life sciences have entered a post-genome era to systematically study gene functions on a large scale[1]. Because of its similarity to humanity in genomic se-quences, biochemical metabolism and physiological mechanism, Mus musculus is the ideal model animal in the study of functional genome. As the publication of the draft map of mouse genome sequences in December 2002, studying gene functions by mouse enters a new stage[2]. So far, there …     

Mapping of genetic modifiers of <Emphasis Type="Italic">Plcd1</Emphasis> in scant hair mice (<Emphasis Type="Italic">snthr</Emphasis><Superscript><Emphasis Type="Italic">1Bao</Emphasis></Superscript>)

The scant hair mutant mouse (locus symbol: snthr ^1Bao ) is a recessive mutation that originated in an ethylnitrosourea chemical carcinogenesis study using the DBA/2J inbred strain. The gene responsible for the mutation was previously determined to be phospholipase C, delta 1 (Plcd1; mutant allele symbol Plcd1 ^snthr1Bao ). To map the modifiers of Plcd1, an intercross (DBA/2J-snthr ^1Bao /snthr ^1Bao × C57BL/6J+/+) was conducted. The F2 mutant progeny exhibited a variety of alopecia phenotypes; all F2 mutants (n=507) were classified into 3 groups (mild, moderate, and severe alopecia) and genotyped based on 96 microsatellites. A major QTL was identified on mouse chromosome (mChr) 15 at 12 cM with an LOD score greater than 7 (P < 0.0001). Three minor QTLs were detected on mChr 2, 5, and 7 at 40, 84 and 48 cM, respectively. The QTLs on mChr 7 and 15 were associated with minor alopecia while the QTLs on mChr 2 and 5 were associated with moderate to severe alopecia. No antagonistic or synergistic effects among or between the 4 QTLs were found. Integrating the functions of the 4 potential regulatory QTLs and mutant Plcd1 ^snthr1Bao , we found that these QTLs might contribute to variations of scant hair severity by altering the Ca²⁺ signal pathways in mouse skin.     

Effect of kinase-negativeEGFR gene on differentiation of embryonic germ cell line EG4

EG4 cells derived from primordial germ cells (PGCs) of 10.5 d post coitum 129/svJ mouse embryos can be used as a model system for in vitro differentiation study due to their pluripotential development ability. EG4 cell lines with stable expression of kinase-negative EGFR cDNA, designated EG4-EGFR^d, were generated by gene transfection. We found that: (ⅰ) EG4-EGFR^d cells share the similar morphology and growing character with wildtype cells that can maintain undifferentiated state in long term culture. (ⅱ) Treatment of EG4 cells with RA resulted in differentiation of adipocyte, while in mutant clones of EG4-EGFR^d, adipocytes were sparse or absent under the same condition, indicating the role of EGFR expressed during adipocyte development. (ⅲ) Histological analysis showed that predominant tissues in teratocarcinomas derived from EG4-EGFR^d cells and wildtype cells are different. A large amount of undifferentiated cells was present in those coming from mutant cell clones. In addition some cardiac and skeletal muscles are prominently differentiated cell types. EG4 wildtype cells produced multiple differentiated cell types of three primary germ layers such as cartilage, epithelia and neural tube. These studies suggested that EGFR-dependent differentiation was inhibited in kinase-negative EG4 cells.     

保护剂浓度对第19期鸡胚原始生殖细胞存活率的影响

采用Ficoll密度梯度离心 ,提取第 19期 (孵化 72h)性腺中的原始生殖细胞 (PGCs) ,用不同浓度的冷冻保护液进行冷冻保存。复苏后的PGCs用台盼蓝染色检测其存活率 ,并进行体外培养。结果发现 :在同一浓度下 ,不同的冷冻保护液之间存在显著 (P <0 0 5 )或极显著 (P <0 0 1)差异。在同一种冷冻保护液下 ,不同的浓度之间存在显著(P <0 0 5 )或极显著 (P <0 0 1)差异 ,具体表现为冷冻保护液浓度越大 ,复苏后细胞的存活率越低。PGCs复苏后于体外培养 ,可增殖形成细胞克隆 ,并可传代培养     

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.     

鸭胚胎血液中PGCs数量及其外源DNA的体内转染

抽取了麻鸭和骡鸭胚胎发育第14－20期的血液，制作血涂片，PAS染色，观察血液中的原生殖细胞的含量，并对各个时期血液中原生殖细胞的含量进行了统计，最高含量都出现在第16期,分别为8．9752％和2．1871％，用In vivo Gene Delivery Systern和pEGFP-N对PGCs进行了外源DNA的体内转染。结果在6只胚胎中的3只中发现GFP报告基因的表达。     

Requirement for mast cell growth factor for primordial germ cell survival in culture.

Mast-cell growth factor (MGF) is encoded by the murine steel (Sl) locus and is a ligand for the tyrosine kinase receptor protein encoded by the proto-oncogene c-kit at the murine dominant white spotting (W) locus. Mutations at both these loci affect mast cells, primordial germ cells (PGCs), haemopoietic stem cells and melanocytes. In many Sl and W mutants, the rapid proliferation of PGC that normally occurs between day 7 and 13.5 of embryonic development fails to occur. As c-kit is expressed in PGCs while MGF is expressed in the surrounding mesenchyme, MGF might promote the proliferation of PGCs. Here we report that MGF is essential for PGC survival in culture, but does not stimulate PGC proliferation. Moreover, whereas both the transmembrane and soluble proteolytic cleavage forms of MGF stimulate mast-cell proliferation, soluble MGF has a relatively limited ability to support survival of PGCs in culture, thus explaining the sterility in mice carrying the steel-dickie (Sld) mutation, which encodes only a soluble form of MGF, and providing a functional role for a transmembrane growth factor.     

Germline stem cells and follicular renewal in the postnatal mammalian ovary

A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.     

半滑舌鳎早期胚胎性腺原基分化的组织学

采用连续组织切片对半滑舌鳎胚胎及仔鱼进行了观察研究,首次描述了半滑舌鳎胚胎发育过程中原始生殖细胞(primordial germ cells,PGCs)出现的部位及迁移特征,以及卵黄合胞体,鳔与性腺原基的发育分化.结果发现,PGCs出现于神经胚期的靠近卵黄囊的囊胚层.PGCs的特征为体积比周围细胞大,核大透亮,随后在肌肉期的脊索壁上出现.孵化前期的PGCs迁移到肠原基附近,肠系膜旁可见尚在移动的PGCs和10日龄的仔鱼中在肾管旁出现性腺原基,以后PGCs数量逐渐增多参与性腺的形成.本研究为半滑舌鳎的发育生物学以及养殖生产实践提供参考.     

鸟类原生殖细胞的实验观察

本文系统地报导了鸟类原生殖细胞(PGC_s)的实验观察。初步提出了关于使用简易方法制备鸟类PGC_s标本的一系列技术(包括取胚、取胚血、制作胚血涂片、活胚标本、整胚装片和胚组织切片等)。实验观察表明,鸟类PGC_s可以在光镜下借助普通细胞学和组织化学相结合的方法进行鉴别。     

Germline transmission of genetically modified primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.     

Embryonic expression of a haematopoietic growth factor encoded by the Sl locus and the ligand for c-kit.

Mice carrying mutations at the W (Dominant white spotting) and Sl (Steel) loci develop abnormalities in three independent systems: neural crest-derived melanocytes, primordial germ cells and haematopoietic stem cells. Consequently, homozygotes of viable mutant alleles have white coats and are sterile and severely anaemic. Tissue recombination studies predict that the W gene is expressed cell autonomously, whereas the product of the Sl locus affects the microenvironment in which the stem cells migrate, proliferate and differentiate. The W locus encodes the protoncogene c-kit, a member of the tyrosine kinase receptor family. The haematopoietic growth factor SCF (stem cell factor) has been identified as the product of the Sl locus and a ligand for c-kit. Here, we report that SCF is expressed during embryogenesis in cells associated with both the migratory pathways and homing sites of melanoblasts, germ cells and haematopoietic stem cells. Both SCF and c-kit are also expressed in a variety of other tissues, including the brain and spinal cord, suggesting that the receptor-ligand system has additional roles in embryogenesis.     

Chromatin dynamics during epigenetic reprogramming in the mouse germ line

A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.     

The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours

In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.     

实验用五指山小型猪胚胎生殖细胞的分离培养

目的对从猪的原始生殖细胞分离获得胚胎干细胞的方法进行初步研究。方法胎儿取自怀孕26～28d的实验用五指山母猪。分离胎儿生殖嵴获得PGCs,接种于STO饲养层上,以DMEM+2mmol/L谷氨酰胺+0.1mmol/L非必需氨基酸+0.1mmol/Lβ-巯基乙醇+100IU/mL青霉素+100μg/mL链霉素+15%FCS作为培养基,对细胞进行培养传代。结果培养的细胞传至4～5代,形态多样,经AKP染色、SSEA-1免疫荧光染色、Oct-4免疫荧光标记等方法鉴定为阳性,具有胚胎干细胞的特征。     

p53 基因敲除小鼠的饲养繁殖及鉴定

摘要: 目的为了繁育和鉴定p53 基因敲除小鼠,将引进的杂合子小鼠进行饲养繁殖,杂合子用于继续保种。方法 对其幼鼠剪尾提取基因组DNA,采用PCR 方法进行基因型鉴定。结果对引进小鼠已成功饲养和繁殖,并得到 纯合基因缺失型小鼠。结论正确的饲养、繁殖及基因鉴定方法对于基因敲除小鼠的获得和保种具有重要的意 义。     

Improvement of Identity-Based Threshold Proxy Signature Scheme with Known Signers

0 IntroductionIdentity-based (ID-based) cryptography[1]is rapidly emer-gingin recent years .The concept of the proxy signature scheme was first in-troduced by Mamboet al[2]in 1996 .The concept of thresholdproxy signature was proposed[3 ,4]. Apractical and secure (t,n) threshold proxy signature scheme should satisfy the secre-cy,the proxy protected,the unforgeability,the non-repudia-tion,the ti me constraint ,andthe known signers[5].In 2006 , Baoet al[6]proposed an ID-based thresholdproxy si…     

静宁鸡原生殖细胞的分离及形态学观察

原生殖细胞(PGCs)是配子的始祖细胞,它可发育演变生成卵细胞或精子细胞.上世纪90年代发展了体外培养PGCs技术,现在PGCs已能通过体外培养形成多潜能干细胞(ES),对于研究体外分化机制和细胞治疗具有重要意义.文章通过对静宁鸡种蛋进行36~82 h孵化,并对其血涂片经PAS染色后,在显微镜下观察其原生殖细胞(PGCs)的形态结构,以便为鸡的胚胎干细胞分离培养奠定基础.实验结果:原生殖细胞呈圆形或卵圆形,明显较其他细胞大2~4倍,PGCs的细胞核颜色较深,偏向一侧,后期常聚成一团.