小鼠精原干细胞的分离、分选、移植和培养

精原干细胞具有自我增殖和分化为精子的能力。精原干细胞的培养、移植和体外诱导分化等研究将使我们最终阐明精原干细胞的自我更新机制和精子发生机理。经过一年多的研究,我们不仅建立了用含血清培养基对分离的乳鼠睾丸细胞进行差异贴壁分选来富集生殖细胞的简单方法,而且成功地对分选出的精原干细胞进行了近一个月的无血清培养。流式分析结果表明,差异贴壁分选能将精原干细胞富集11倍以上。移植分析表明,分选出的精原干细胞具有在受体鼠的曲精小管中产生克隆的能力和正常生精作用。免疫细胞化学和RT-PCR检测表明,培养的细胞表达精原干细胞标志基因。该研究为体外长期培养小鼠或其它哺乳动物的精原干细胞提供了一个范例,也为利用基因修饰的精原干细胞通过移植产生转基因动物奠定了基础。     

Germ cell transplantation in infertility mouse

This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules. Donor germ cells were isolated from male FVB/NJ-GFP trensgenic mice. Seminiferous tubule microinjection was applied to achieve intratubular germ cell transfer. The germ cells were injected into exposed testes of the infertility mice. We used green fluorescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker. The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjection. The spermatogenesis was morphologically observed from the seminiferous tubules in 41/60 （68.33%） of the injected recipient mice using allogeneic donor cells. In the colonized testes, matured spermatozoa were seen in the lumen of the seminiferous tubules. In this research, BALB/c-nu infertility mouse model, the recipient animal, was used to avoid immunological rejection of donor cells, and germ cell transplantation was applied to overcome infertility caused by busulfan treatment. These results demonstrate that this technique of germ cell transplantation is of great use. Germ cell transplantation could be potentially valuable to oncological patients.     

Prospect of creating transgenic animals by using spermatogonial transplantation

Transgenic animal technology is a powerful tool for researching bioscience, biomedicine, bioreactor, and agriculture. There are various ways to produce transgenic animals. The most common ways currently available are pronuclear microinjection and nuclear transfer techniques. However, these methods usually result in low efficiency, causing mosaic （in pronuclear microinjection）, or developmental abnormalities （in nuclear transfer）. In 1994, Brinster and his colleagues reported an original method to transfer spermatogonial stem cells from donor to recipient mice. The donor spermatogonia were able to form spermatozoa in recipient testes, and to produce progeny carrying the donor＇s genetic characters. Since then, a series of novel methods were invented by using spermatogonia transplantation. These new methods facilitate the research and application of spermatogonia. Some of these methods, when combining with genetic modification methods, will form a novel methodology for creating transgenic animals. The present paper reviews the achievements of research on spermatogonia transplantation related to creating transgenic animal. Such as, transplantation techniques, cryopreservation of spermatogonia, preparation of recipients, long-term proliferation of spermatogonia in culture, genetic modification of spermatogonia, and characterization of germ line transmission of the modified gene, etc. Furthermore the methodologies for creating transgenic animals by using spermatogonia transplantation were described. Based on the difference between donors and recipients used, the methodology is categorized into two groups： allogeneic transplantation, and autologous transplantation. Although progress in this research area has been swift, potential difficulties remain to be overcome in each approach. The advantages and existing problems in the methodology are discussed.     

花粉管通道法转基因技术在甜瓜品种河套蜜瓜上的应用

为建立甜瓜(Cucumis melo L.)简便易行转化频率高的转基因技术,以甜瓜品种河套蜜瓜为受体材料,以含gus基因的植物双元表达载体pPZP221为外源基因供体,进行了花粉管通道法转基因研究.自交授粉后分别于1、2、3、4、5、6、7、8、9、10 h,切去柱头上端1/3部分,立即滴加质粒DNA溶液,果实成熟后收获转化种子.对T0代植株进行PCR检测,结果表明不同时间处理所得到的T0代植株均具有较高的转化频率,其中授粉后7 h滴加DNA溶液所获得的转化率为28.3%.对一部分PCR阳性的T0代植株基因组DNA进行Southern杂交分析,结果表明所有样品均出现特异性杂交条带,证明外源基因已整合到受体植物基因组中.     

Genotyping of Rhesus SCNT pluripotent stem cell lines

Somatic cell nuclear transfer (SCNT) into enucleated oocytes has emerged as a technique that can be used to derive mouse embryonic stem cell lines with defined genotypes. In this issue Byrne et al. report the derivation of two SCNT Rhesus macaca male stem cell lines designated CRES-1 and CRES-2. Molecular studies detailed in their paper provides supporting evidence that the chromosome complement of CRES-1 and CRES-2 was genetically identical to the male cell donor nucleus and that the mitochondrial DNA originated from different recipient oocytes. In this validation paper, we independently confirm that both stem cell lines were indeed derived by SCNT.     

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the nuclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation ogene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.     

造血重建小鼠骨髓中成纤维细胞的起源

选择Y染色体特异的性别决定基因(Sry)作为新的细胞遗传标志,采用PCR技术对小鼠骨髓细胞重建造血的性能和造血重建小鼠骨髓中成纤维细胞的起源进行研究.将正常雄鼠骨髓细胞输注给经致死剂量射线照射的受体雌性小鼠,PCR技术检测结果表明,在活存小鼠的骨髓、脾脏、胸腺和淋巴结中均具有供体起源的细胞.而正常雄鼠或5-氟尿嘧啶处理的雄鼠骨髓细胞输注给受体雌性小鼠后,造血重建小鼠骨髓中的成纤维细胞则为受体起源.由此可见,小鼠骨髓中的成纤维细胞与造血细胞具有不同的起源.     

富钾植物DNA导入早稻变异后代的RAPD分析

应用花粉管通道法和浸种法将两种富钾植物空心莲子草和商陆DNA导入到不同的水稻品种中,结果在其后代产生了广泛的变异,并从中筛选出耐低钾的变异材料。应用PCR技术对其中耐低钾的4个变异材料进行了RAPD分析。结果表明,所用100个随机引物中有7个引物在受体和变异后代间扩增出了多态性产物。后代和受体的相似系数均在90%以上,而与供体的相似系数只有6%以下,其中有三个引物的扩增产物中出现了供体的特异性带。这说明空心莲子草和商陆DNA导入水稻后确实引起了水稻基因组结构的变化,同时也为变异后代所发生的生物学性状的改变提供了直接的分子证据。     

扩频通信在数据传输中的应用

以扩频通信发展现状为背景,借助Matlab通信仿真技术和信道编码相关理论知识,对扩频通信和信道编码的相关技术问题进行了分析和研究,提出解决无线通信传输中干扰的方案;选择了（7、4）汉明码、（2、1、3）卷积码和级联码作为研究的对象,分别从理论上和硬件上进行了测试。     

长江口及其邻近海域洪季悬沙分布特征分析

基于近几年大量的实测资料,对长江口及其邻近海域洪季期间悬沙浓度的时空分布特征进行了分析.结果表明,悬沙浓度空间上以杭州湾测点最高,其次是长江口内、长江口外,舟山海域最低;悬沙平面分布总体态势为近岸高而外海低;长江口纵向上自口内经口门向口外悬沙浓度大致呈“低-高-低”分布,沿杭州湾测点则由西向东逐渐降低;横向比较长江口外海滨四个测点发现其悬沙浓度自北向南顺次升高;悬沙浓度一般由表及底逐渐增大,但在不同水域其主要呈现的垂线结构类型则有所不同.时间上由于潮流的大小潮和潮周期变化,悬沙浓度还存在大小潮和涨落潮周期变化.另外,对长江口及其邻近海域悬沙浓度时空分布特征的原因进行了初步分析.     

Tet-on系统诱导乳腺特异表达CUEDC2转基因小鼠的制备

目的：建立Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠模型。方法：构建转基因表达载体,经细胞诱导表达验证后,酶切得到含有CUEDC2的线性DNA片段,并采用显微注射的方法及后续筛选鉴定,得到CUEDC2转基因小鼠。进而通过与乳腺特异表达的MMTV-rtTA转基因小鼠交配,得到CUEDC2/rtTA双阳性转基因小鼠。利用2mg/ml的多西环素（Doxycycline）诱导小鼠体内CUEDC2表达,通过Western Blot、免疫组化检测表达。结果：经诱导,CUEDC2/rtTA双阳性转基因小鼠的2个Founder的F1、F2代在转录水平和蛋白水平均成功表达CUEDC2, 并具有乳腺特异性。结论：Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠制备成功.     

基因枪法转基因技术的研究综述

转基因技术是将目的基因导入受体中,使该基因在受体生物中得以表达．基因枪法是一种重要的基因转化方法．论述了基因枪技术产生的历史背景、基因枪技术的转化原理、影响基因转换效率的因素以及基因枪技术的应用和发展前景等．     

苜蓿转基因研究进展

植物遗传转化是指利用分子生物学的手段将外源基因导入受体植物,使其获得新的性状.传统的育种方法进展缓慢,周期长,而且短期内性状表现不明显.转基因技术比传统的育种方法能快速有效地定向培育植物新品种,获得的新性状比较明显.因此,它已成为植物育种研究领域热点.目前常用的植物遗传转化法包括农杆菌介导,基因枪直接转化等方法,但农杆菌介导法由于外源基因整合的拷贝数少,重排程度低,转化效率高,是最常用的苜蓿遗传转化法.现在通过农杆菌介导法已获得了抗逆,抗除草剂和品质改良等新性状转基因苜蓿,为苜蓿遗传育种开辟了一条新的途径.     

克隆西农莎能奶山羊的微卫星DNA分析

目的利用微卫星DNA技术对通过体细胞克隆技术获得的两只西农莎能奶山羊进行鉴定。方法提取两只克隆西农莎能奶山羊、两只受体西农莎能奶山羊母羊、西农莎能奶山羊供体细胞和两只对照组西农莎能奶山羊母羊的基因组DNA,通过5对具有显著多态性差异的引物扩增微卫星DNA序列,并对其进行微卫星DNA分析。结果两只克隆西农莎能奶山羊和供体细胞的基因型完全一致,受体母羊和对照组母羊的微卫星DNA多态性则与前两者均不相同。结论两只克隆西农莎能奶山羊基因组DNA来源于供体细胞。     

基于堆叠自编码器神经网络的复合电磁检测铁磁性双层套管腐蚀缺陷分类识别方法

铁磁性双层套管长期服役于恶劣的工作环境,极易出现腐蚀缺陷,定期为服役中的双层套管进行在线检测十分必要,而对管壁腐蚀缺陷位置的分类识别是管道定量检测与维修的前提和基础,实时准确的套管腐蚀缺陷分类识别能力是决定管道在线检测效率的重要因素。针对这一情况,将脉冲远场涡流和脉冲涡流技术相结合,提出了基于堆叠自编码器神经网络的分类方法。通过仿真和实验选取合适特征量作为输入层,实现了内管外壁腐蚀、外管内壁腐蚀和外管外壁腐蚀的分类,实验整体预判精度可达97.5%,结果表明该方法可对双层套管腐蚀缺陷缺陷实施高效、高精度分类识别。     

2L支路SSC-MRC混合分集及其性能分析

分集合并技术是对抗多径衰落的有效手段,而常见的单纯分集合并技术如最大比合并(MRC)、等增益合并(EGC)、选择合并(SC)以及切换驻留合并(SSC)等在系统复杂度和系统性能之间存在矛盾;所以有必要寻找一种工程易于实现同时性能也可以接受的混合分集合并方式.在此将低成本、结构简单的SSC分集与最优性能的MRC分集结合,即SSC-MRC混合分集合并技术.推导了2L条独立同分布瑞利衰落信道下SSC-MRC混合分集的输出信噪比概率密度函数(PDF)以及BPSK信号下误码率表达式,还分析了门限选取对系统的影响.仿真实验表明,四支路SSC-MRC混合分集性能稍差于四支路MRC,但重要的是节省了2台接收机,大大减少了成本和系统复杂度.     

石蜡包埋组织芯片制作的质量控制研究

目的:通过构建不同规格的石蜡包埋组织芯片,探讨在制作石蜡包埋组织芯片过程中质量控制的关键要素。方法:利用组织点样仪构建组织微阵列蜡块,按照常规方法进行制片,分别进行HE染色与免疫组织化学染色,光镜下检测组织芯片的制作质量。结果:组织微阵列蜡块中的供体蜡块组织排列整齐,组织芯与受体蜡块融合致密,组织芯片切片基本完整,厚薄均匀。经HE染色和免疫组织化学染色后组织形态可利用率在64.04%~93.75%。结论:良好的供体蜡块与受体蜡块、准确的组织定位、合适的排列方式和熟练的组织芯片制备技术是构建高质量组织芯片的关键因素。     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

一个药用野生稻异源单体附加系在减数分裂时期的GISH分析鉴定

异源单体附加系是从亲缘关系较远或属间的一个物种单条染色体附加到另一个物种中．栽培稻珍籼97B与药用野生稻Hy18杂交与连续回交，在BC2后代中得到一个药用野生稻单体附加系．生物素标屺的药用野生稻总DNA作为探针，未标记的栽培稻总DNA封阻，对其异源单体系减数分裂染色体进行基因组原位杂交．FISH结果表明，在栽培稻（AA，2n=24）基因组中附加了一条药用野生稻染色体，并鉴定为第8号染色体．研究表明，药用野生稻异源单体附加系的建立为药用野生稻的基因组学和遗传学的研究提供一个新的操作平台．而GISH技术在水稻远缘杂交育种中是最准确有效的染色体鉴定方法．在水稻育种改良中具有重要应用前景．     

Effects of sub-lithospheric small-scale convection with a Newtonian rheology on the seafloor topography and heat flux

Scafloor topography and heat flux show clear dependence on the age of seafloor. A half-space cooling (HSC)model can reproduce seafloor topography and heat flux data for younger seafloor, but for older seafloor the observations show reduced variations with the age in comparison with the HSC model predictions. The deviation was attributed to the sub-lithospheric small-scale (SSC) convection first by Parsons and McKenzie (1978). While there is little doubt that the SSC can enhance heat flux at relatively old seafloor, questions were raised as to whether or not the SSC can actually lead to a reduced topography. In this study, the effects of SSC on scafloor topography and heat flux are investigated by formulating a 2-D thermal convection model that is parallel to plate motion. Instead of using closed boundary conditions,which will bring large pressure effects because of return flow,a flow through boundary condition is adopted. The results show that although the SSC enhances the surface heat flux, it has little effects on topography for the fluids with a more realistic rheology. The reason for this is that the SSC transports the heat from the bottom to the top and cools down the whole fluids, and with the existence of a stagnant lid, the whole effects on topography are negligible.