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.     

胡萝卜与川西獐牙菜不对称体细胞杂交

川西獐牙菜(Swertia musstii Franch)原生质体经260μw／cm^2紫外线照射30s、1min、2min、3rain后，与胡萝卜(Daucus carota L.var.sative DC)原生质体在PEG诱导下融合．融合再生的110个单细胞克隆形态学、染色体、同功酶分析表明，35个含有酯酶及／或过氧化物酶同工酶的双亲特征带，部分杂种产生新带，染色体数目多为16-18，确证它们为体细胞杂种．     

醉蝶花的组织培养及其细胞遗传学研究

研究发现赤霉素对醉蝶花(Cleome spinosa Jacq.)种子发芽和幼苗生长有明显的促进作用，其中10mg／L浸种30min的处理对种子发芽和幼苗生长的促进作用最明显，发芽率达到80％．用不同激素组合诱导醉蝶花下胚轴和子叶形成愈伤组织，MS 6-BA0．5mg/L NAA0．5mg/L KT0．5mg／L中子叶的出愈率最高，达到100％．下胚轴出愈不及子叶，最高只有93％．MS 6-BA2mg／L NAA0．05mg/L适合于芽的分化，诱导率达到100％．以醉蝶花幼芽为材料，用常规染色体制片方法确定醉蝶花体细胞染色体数目2n=20．     

小麦体细胞胚发生中蛋白质组分和过氧化物酶同工酶的变化

用PAGE及薄层找扫描方法分析了小麦体细胞胚发生和发育的不同阶段蛋白质组分和过氧化物酶酶谱的变化。胚性愈伤组织,从球形胚、梨形胚,盾片胚到成熟胚不同发育时期蛋白质电泳图谱相似,与对照(非胚性愈伤组织)明显不同。前者有18KD,49KD,78KD的蛋白质组分,其含量随发育进程而上升。对照则无这些蛋白质组分或含量很低,但含有特异的14KD蛋白质。胚性愈伤组织和体细胞胚的不同发育时期过氧化物酶同工酶酶谱极其相似,都具有C_2、C_6、C_93条特异酶带,但活性有差异。对照不存在这3条酶带。再生苗的蛋白质电泳图谱和过氧化物酶同工酶酶谱介于胚性和非胚性组织之间。     

玉米幼胚愈伤组织再生植株的变异

玉米受粉7—10天的幼胚,在含激素的MS培养基上诱导生成愈伤组织,并分化出胚状体,成苗后移栽于温室内,至成熟结实。这些试管苗植株在整个生长发育过程中,出现了雌穗顶生,顶生雌雄同穗,植株矮,叶片少,雌穗着生部位低及轮生叶序和对生叶序等多种显著的差异和变异。     

聚乙二醇诱导细胞融合影响因素探究

以鸡红血细胞为材料,聚乙二醇(Mr=4 000)为诱导剂,研究不同的细胞密度、聚乙二醇体积分数、Ca2 浓度等因素对细胞融合率的影响,结果显示:细胞密度为(3.0~4.0)×104/mL,聚乙二醇的体积分数为50%,Ca2 浓度为50mmol/mL时融合效果最好,融合率高达29.76%.     

Analysis of OCT4 expression in an extended panel of human tumor cell lines from multiple entities and in human mesenchymal stem cells

OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrowderived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature. Received 30 September 2008; received after revision 05 November 2008; accepted 10 November 2008     

AID in antibody perfection

Expressed immunoglobulin (Ig) genes undergo alterations in sequence and genomic structure in order to optimize antibody function. A single B cell-specific factor, activation-induced deaminase (AID), initiates these changes by deamination of cytosine to uracil. Uracil in DNA is encountered commonly, and conserved pathways are responsible for its faithful repair. However, at the Ig loci of B cells, AID-initiated damage is processed to produce three distinct outcomes: somatic hypermutation, class switch recombination and gene conversion. This review focuses on the role of AID in Ig gene diversification, emphasizing how AID functions within the mechanism of the Ig gene diversification pathway; and highlights open questions for future research, particularly the most provocative current question: what makes a gene a target for AID-initiated mutagenesis?     

甘薯胚性悬浮系的建立及其细胞生长特性研究

徐薯18叶片接种在附加2mg/L,4-D的MS固体培养基上，形成了质地松散的胚性愈伤组织，胚性愈伤组织在附加1mg/L,2,4-D的MS液体培养基中进行悬浮培养，建立了增殖迅速、分散度良好的具有较高体胚分化能力的细胞悬浮系。小型细胞团（直径小于350μm）以2％－4％的接种量转移到无激素MS液体培养基中，可分化出大量体胚。在继代初期，悬浮系pH值呈下降趋势，细胞处于旺盛分裂时期，导致悬浮系鲜质量、干质量、细胞紧实体积（PVC）的显著升值；4d以后，pH值逐渐回升，细胞分裂缓慢而体积增长迅速，导致鲜质量、PVC增加而干质量少有变化；6d以后pH值继续回升而细胞分裂和生长受到明显抑制。     

Molecular basis and genetic improvement of economically important traits in aquaculture animals

Aquaculture has been believed to be a major Chinese contribution to the world. In recent 20 years, genome and other genetic technologies have promoted significant advances in basic studies on molecular basis and genetic improvement of aquaculture animals, and complete genomes of some main aquaculture animals have been sequenced or announced to be sequenced since the beginning of this century. Here, we review some significant breakthrough progress of aquaculture genetic improvement technologies including genome technologies, somatic cell nuclear transfer and stem cell technologies, outline the molecular basis of several economically important traits including reproduction, sex, growth, disease resistance, cold tolerance and hypoxia tolerance, and present a series of candidate trait-related genes. Finally, some application cases of genetic improvement are introduced in aquaculture animals, especially in China, and several development trends are highlighted in the near future.