Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development

Introduction of selectable marker genes to transgenic animals could create an inconvenience to further research and may exaggerate public concerns regarding biological safety. The objective of the current study was to excise loxP flanked neo^R in transgenic cloned cattle by transient expression of Cre recombinase. Green fluorescent protein gene (GFP) was incorporated to monitor Cre expression; therefore, Cre-expressed cells could be selected indirectly by fluorescence-activated cell sorting (FACS). The neo^R was removed and Cre expressed transiently in GFP-positive colonies; excision of neo^R was confirmed by single-blastocyst PCR in recloned blastocysts, with neo^R-free fibroblast cells as donors. There was no difference (P > 0.05) in rates of cleavage (76.0% vs. 68.8%) or blastocyst formation (56.6% vs. 52.9%) between recloned embryos with neo^R-free or neo^R-included donors. The differential staining of recloned blastocysts were similar (P >0.05) in terms of total cell number (124 vs. 122) and the ratio of ICM (Inner Cell Mass) to the total cell number (38.1% vs. 38.2%). Furthermore, pregnancy and calving rates were not different (P > 0.05) from those of the control. In conclusion, we successfully excised neo^R from transgenic cloned cattle; the manipulation did not affect the developmental competence of recloned preimplantation embryos. This approach should benefit bioreactor and transgenic research in livestock.     

陆地棉品种抗黄萎病反应规律的研究

对我国自育的108个陆地棉品种的抗黄萎病性进行了研究。在黄萎病发病期内,对黄萎病发病情况进行连续调查,测定产量、考查产量因素并检测纤维品质。利用因子分析法对陆地棉抗黄萎病反应规律进行分析,得出不同时期的黄萎病病指主要与前后3～5个阶段抗病性有关。病情发展主要由4个主因子决定,且第1、2主因子具有较大的方差贡献率。第1主因子(F1)主要与品种7月26日至8月9日的黄萎病病指有关,第2主因子(F2)主要与品种8月20日至9月4日的黄萎病病指有关。利用因子分析结果将108个品种划分为4个类型,前期抗病性较好而后期发病较快的第Ⅰ类品种,其产量较低,单株结铃数、单铃重、衣分均低于其他3类;纤维品质均较差,纤维长度、整齐度、比强度和马克隆值均较其他3类差。前期和后期病指均较低、发病缓慢的第Ⅱ类则小区产量最高,纤维品质处于平均水平。第Ⅲ类品种前期发病较慢,中期发病较快,具有较高的小区产量,单铃重最高;纤维整齐度、比强度和伸长率好于其他3类品种;前期发病较快,中期发病平缓,后期仍具有较高病指的为第Ⅳ类品种,小区产量较低,单株产量、单株结铃数和衣分较高;其他性状处于中等水平。但研究表明,某一阶段具有的抗病性并不能完全代表品种的抗病性。     

昆虫保幼激素促进家蚕杆状病毒系统的基因表达

杆状病毒表达载体系统(Baculovirus Expression VecterSvstem,BEVS)的一个最大优点是外源基因的高效表达(Hy-perexpression).但是,不同的外源基因在BEVS系统中的表达水平相差很大,较低的如α-干扰素,表达量为1～5mg/L培养细胞;高的如β-半乳糖苷酶,表达量可达600mg/L培养细胞.外源基因在BEVS系统中表达量受到诸多因素的影响,如细胞的类型与质量,外源基因蛋白的性质,启动子序列的完整性,是否为融合蛋白等[1].如何使外源基因在BEVS系统中高效表达,是近年来该领域中研究最活跃的方向之一.已证实家蚕杆状病毒的表达量受宿主遗传型的影响,最低和最高的遗传型相差达7倍以上[2].林水中等发现家蚕饲料中添食适当浓度的硫酸铜可提高外源基因单位表达量10%左右[3].杆状病毒在复制循环中表现出两种类型:芽生病毒和包涵体病毒,其中芽生病毒引起宿主体内不同组织间的感染,包涵体病毒则引起宿主之间感染[1].杆状病毒基因组中蜕皮激素尿苷二磷酸葡萄糖基转移酶(egt)基因影响激素在宿主体内的平衡[4],egt基因通过糖基化作用使蜕皮激素失活,打破宿主体内的激素平衡,延长幼虫期,以利于病毒的增殖[5].家蚕血淋巴中保幼激素(Juvenile hormone,JH)的滴度同样决定着幼虫发育的进程[6],本文通过体表使用保幼激素,以研究保幼激素对家蚕核型多角体病毒和宿主之间的相互关系及对外源基因表达量的影响.     

Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum cells to the anticancer drug cisplatin

The drug cisplatin is widely used to treat a number of tumor types. However, resistance to the drug, which remains poorly understood, limits its usefulness. Previous work using Dictyostelium discoideum as a model for studying drug resistance showed that mutants lacking sphingosine-1-phosphate (S-1-P) lyase, the enzyme that degrades S-1-P, had increased resistance to cisplatin, whereas mutants overexpressing the enzyme were more sensitive to the drug. S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine kinase. We have identified two sphingosine kinase genes in D. discoideum--sgkA and sgkB--that are homologous to those of other species. The biochemical properties of the SgkA and SgkB enzymes suggest that they are the equivalent of the human Sphk1 and Sphk2 enzymes, respectively. Disruption of the kinases by homologous recombination (both single and double mutants) or overexpression of the sgkA gene resulted in altered growth rates and altered response to cisplatin. The null mutants showed increased sensitivity to cisplatin, whereas mutants overexpressing the sphingosine kinase resulted in increased resistance compared to the parental cells. The results indicate that both the SgkA and the SgkB enzymes function in regulating cisplatin sensitivity. The increase in sensitivity of the sphingosine kinase-null mutants was reversed by the addition of S-1-P, and the increased resistance of the sphingosine kinase overexpressor mutant was reversed by the inhibitor N,N-dimethylsphingosine. Parallel changes in sensitivity of the null mutants are seen with the platinum-based drug carboplatin but not with doxorubicin, 5-fluorouracil, and etoposide. This pattern of specificity is similar to that observed with the S-1-P lyase mutants and should be useful in designing therapeutic schemes involving more than one drug. This study identifies the sphingosine kinases as new drug targets for modulating the sensitivity to platinum-based drugs.     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

新疆罗布麻生态类型及其纤维品质研究

新疆塔里木河及叶尔羌河流域是我国能够提供商品精干罗布麻的主要地区。由于野生罗布麻生长高矮不一,形态各异,与其纤维品质的相关性较大。通过40个株号的罗布麻植株形态和纤维长度等的的测定,分析各类型罗布麻的纤维长度和罗布麻植株各部分纤维长度情况。研究结果表明：罗布红麻高杆类型主茎纤维最长,罗布麻放牧类型纤维最短。罗布麻植株各部分纤维长度是主茎上的大于分枝,主茎中部的最长,基部和梢部最短。为野生罗布麻资源开发利用提科学依据。     

Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice

The vacuole, a multifunctional organelle of most plant cells, has very important roles in space filling, osmotic adjustment, storage and digestion. Previous researches suggested that aquaporins in the tonoplast were involved in vacuolar functions. The rice genome contains 33 aquaporin genes, 10 of which encode tonoplast intrinsic proteins (TIPs). However, the function of each individual TIP isoform and the integrated function of TIPs under various physiological conditions remain elusive. Here, five rice TIP members were characterized with water and/or glycerol transport activities using the Xenopus oocyte expression system. OsTIP1;2, OsTIP2;2, OsTIP4;1 and OsTIP5;1 possessed water transport activity. OsTIP1;2, OsTIP3;2 and OsTIP4;1 were demonstrated with glycerol transport activity. Rice TIP expression patterns under various abiotic stress conditions including dehydration, high salinity, abscisic acid (ABA) and during seed germination were investigated by real-time PCR. OsTIP1s (OsTIP1;1 and OsTIP1;2) were highly expressed during seed germination, whereas OsTIP3s (OsTIP3;1 and OsTIP3;2) were specifically expressed in mature seeds with a decrease in expression levels upon germination. The results of this research provided a functional and expression profiles of rice TIPs.     

生物转化生产D-塔格糖的食品级菌种选育及L-阿拉伯糖异构酶的克隆表达

L-阿拉伯糖异构酶(L-arabinose isomerase,L-AI)是一种可以催化D-半乳糖为D-塔格糖的胞内异构化酶。随着塔格糖在食品工业中越来越广泛的应用,能够将半乳糖转化为塔格糖的食品级微生物以及食品级来源的L-AI受到更大的关注。文中从各种酸奶制品、泡菜及其他一些食品中采集不同的样品,筛选出1株具有L-AI酶活的食品级菌株,经过生理生化鉴定以及16S rDNA序列测定,确定该菌株为戊糖片球菌,命名为Pediococcus pentosaceus PC-5。以该菌基因组为模板,克隆L-AI基因,并在大肠杆菌BL21成功地异源表达。表达产物经粗提取后,在40℃下加入Mn2+,使D-半乳糖转化为D-塔格糖的转化率为33%。