四倍体补偿技术的建立及其应用

常规基因剔除小鼠的获得主要是利用ES细胞的全能性先获得嵌合体小鼠,再利用:ES细胞的生殖系传递能力,通过嵌合体与野生型小鼠的交配获得杂合子小鼠.而四倍体补偿技术则可绕过嵌合体小鼠阶段,直接获得基因修饰杂合子小鼠.利用电融合技术和Piezoelectric microinjecfion显微注射技术建立了四倍体补偿技术,小鼠四倍体胚胎的获得率(电融合率)为(93.01±l.37)%,经体外培养囊胚形成率为(82.49±2.08)%.通过显微注射方法将2种129品系小鼠来源的ES细胞(CJ7和SCR012)注射到四倍体囊胚腔中,获得了完全ES细胞来源的小鼠,ES鼠的获得率分别为2.7%和8.3%.经微卫星DNA检测,成体小鼠的10个被检测组织均为129小鼠来源的.同时,也利用基因修饰的ES细胞进行了研究,获得了2种基因修饰的完全ES细胞来源的杂合子小鼠,部分小鼠具有繁殖能力,经繁育已获得了纯合子,其中凝血因子Ⅷ基因敲除小鼠获得了预期的血友病小鼠表型.上述结果说明四倍体补偿技术可应用于基因修饰小鼠的制备.     

获得多能性干细胞的方法

胚胎干细胞系的获得为细胞和器官损伤及病变的治疗提供了新的途径,但是治疗用细胞和受体病人之间免疫不相容问题妨碍了干细胞临床应用.近年来对分化细胞重编程研究使研究人员可以获得多能性干细胞,这为解决这一难题带来了新的希望.对获得多能性干细胞所涉及的机制以及方法进行了综述.     

产蛋白酶毛霉菌株的初步筛选

对从农家自制豆酱中分离获得的 26 株毛霉菌株,采用酪素培养基平板透明圈法进行产蛋白酶活性初筛,获得了高产蛋白酶活力的菌株M-339、M-513和M-808.改变培养温度和培养基配比,采用Folin-酚法测定蛋白酶活力,分别获得了菌株M-339、M-513和M-808产蛋白酶活力的最佳培养条件.筛选出的菌株有望用于发酵食品的工厂化生产.     

肥胖基因的分离及其在大肠杆菌中的表达

利用PCR技术自外周血白细胞染色体DNA中扩增获取了肥胖基因(ob基因)的外显子2和3序列.经过拼接,获得了全长的ob基因编码序列. 测序结果表明,获得的序列与文献报道完全一致.利用PCR技术扩增出成熟蛋白的编码序列,克隆至表达载体pBV220中获得了表达菌株,并对表达产物进行了初步纯化,为进一步研究ob基因产物的功能与应用奠定了基础.     

民猪的SLA-DRB基因的PCR-RFLP多态性分析

采用克隆测序和PCR-RFLP相结合的方法,对民猪的SLA-DRB基因的整个编码区进行了扫描和多态性分析.结果表明该基因的第2外显子是整个基因的高变区,突变率达到5.6%,其中80%为有效突变,但没有发现新的等位基因;PCR-RFLP结果表明外显子1用内切酶Alu Ⅰ酶切可获得3种基因型;外显子2用内切酶Taq Ⅰ酶切可获得3种基因型;外显子3用内切酶Bcn Ⅰ酶切可获得3种基因型;外显子4用内切酶Mbo Ⅰ酶切可获得2种基因型;外显子5用内切酶Hin1 Ⅰ酶切可获得3种基因型.Hardy-Weinberg平衡分析表明民猪在Alu Ⅰ和Hin1 Ⅰ处于平衡状态(P＞0.05),在Taq Ⅰ、Bcn Ⅰ和Mbo Ⅰ处于不平衡状态.     

格特隐球菌HOG1基因的敲除及重建

目的 构建格特隐球菌HOG1基因缺陷株和HOG1基因重建株.方法 从格特隐球菌基因组扩增HOG1基因,通过部分基因缺失方法,获得缺陷基因dHOG1.将获得的HOG1基因及其缺陷基因dHOG1分别亚克隆到真核表达载体pGAPzα-A,构建pGAPzα-HOG1及pGAPzcα-dHOG1质粒.将pGAPzα-dHOG1质粒转染隐球菌原始株,通过筛选获得HOG1基因缺陷菌株;同样方法将pGAPzα-HOG1质粒转染格特隐球菌HOGI基因敲除菌株,获得HOGI基因重建株.结果 RTPCR结果示:格特隐球菌HOG1基因缺陷株不转录表达完整的HOG1基因,而HOG1基因重建株可以转录表达完整的HOG1基因片段.结论 成功获得格特隐球菌HOG1缺陷株和HOG1基因重建株,为后续格特隐球菌毒力和致病机制的研究奠定了基础.     

广西甘蔗根际高效联合固氮菌的筛选及鉴定

对广西主要甘蔗产区的根际联合固氮细菌进行了收集和评价,拟筛选获得对甘蔗具有潜在促生性能的联合固氮菌,为甘蔗生产节肥减耗提供依据。结合nifH基因扩增和固氮酶活性分析方法筛选获得36个固氮细菌菌株;进一步对所获得固氮菌株的固氮能力、溶磷性、分泌植物生长素IAA的特性等促进植物生长潜能进行评价,获得了5个同时具有较强固氮能力、降解无机磷和分泌植物生长激素IAA的功能菌株;通过Biolog鉴定系统和16S rRNA序列分析对5个具有较好应用潜力的固氮菌进行分类鉴定。结果表明这5个菌株分别属于Klebsiella sp.、Bacillus megaterium、Pseudomonas sp.、Pantoea sp.和Burkholderia sp.。本研究结果表明广西甘蔗根际联合固氮菌具有较大的开发利用潜力。     

BPOZ基因剔除小鼠模型的建立

BPOZ是在卵巢癌等肿瘤组织中表达下调的细胞生长抑制基因,建立BPOZ基因剔除小鼠模型,可以为在体研究BPOZ基因的生物学功能及其与肿瘤发生的关系创造条件.运用生物信息学手段确定小鼠BPOZ基因组序列,设计基因剔除策略,构建完成了基因剔除载体XpPNT-BPOZ.以电穿孔方法将基因剔除载体导入ES细胞,用G418和Ganciclovoir进行正负筛选,获得抵抗克隆,PCR和DNA印迹鉴定出正确同源重组的ES细胞克隆.将同源重组的ES细胞注入小鼠囊胚,获得嵌合体小鼠.嵌合体小鼠与C57BL/6J小鼠交配后获得Aguoti毛色的小鼠30只,其中15只为BPOZ基因剔除杂合子小鼠,阳性率为50%.在雌雄杂合子交配的后代中获得纯合子小鼠.初步的表型观察发现BPOZ基因剔除小鼠发育正常,有繁殖能力,进一步的表型分析工作正在进行之中.     

P-糖蛋白的表达及其结合肽的筛选

为获得P 糖蛋白胞外段 ,构建了高效表达载体pGEX Pgp ,转化大肠杆菌DH5α ,进行表达、鉴定及纯化 ,以获得的融合蛋白为靶蛋白 ,筛选噬菌体随机 12肽库 ,免疫细胞化学方法进行鉴定 .SDS PAGE分析 ,表达出约 30kD大小的蛋白 ;从噬菌体随机肽库中筛选获得了与P 糖蛋白特异性结合的噬菌体阳性克隆 ,测序获得了其特异性结合肽序列 :NDGLLFTWQPSP .免疫细胞化学结果显示 :筛选得到的噬菌体阳性克隆可与耐药细胞BIU 87 ADM结合 ,而与敏感细胞BIU 87不结合 .结果表明 ,筛选获得的结合肽可与耐药的肿瘤细胞结合 ,表现出一定的肿瘤特异性 .P 糖蛋白结合肽的筛选 ,为进行人膀胱癌多药耐药的靶向治疗等工作奠定了基础 .     

以P型职PRSV-VPg为模板多位点突变获得W型PRSV-VPg的研究

以P型PRSV-VPg(Papaya Ringspot Virus,VPg gene)基因为模板利用高保真酶通过两对引物(突变4个位点),PCR扩增获得一条276 bp和一条141 bp的两个小片段.以此为基础,利用长引物延伸和套叠PCR的方法,定点突变剩余5个位点,最后拼接获得W型PRSV-VPg基因.结果表明通过上述方案可以成功改造P型PRSV-VPg基因,获得与W型朋PRSV-VPg同源的目的基因,成功率为100%.该技术可以绕开传统的通过寻找毒源进行RT-PCR方法和人工合成方法获得病毒基因,其技术操作简单,且节约时间和成本,并在人工合成基因、多位点基因定点突变等方面具有很好的借鉴作用和应用价值.     

Mean square stability of uncertain stochastic BAM neural networks with interval time-varying delays

The robust asymptotic stability analysis for uncertain BAM neural networks with both interval time-varying delays and stochastic disturbances is considered. By using the stochastic analysis approach, employing some free-weighting matrices and introducing an appropriate type of Lyapunov functional which takes into account the ranges for delays, some new stability criteria are established to guarantee the delayed BAM neural networks to be robustly asymptotically stable in the mean square. Unlike the most existing mean square stability conditions for BAM neural networks, the supplementary requirements that the time derivatives of time-varying delays must be smaller than 1 are released and the lower bounds of time varying delays are not restricted to be 0. Furthermore, in the proposed scheme, the stability conditions are delay-range-dependent and rate-dependent/independent. As a result, the new criteria are applicable to both fast and slow time-varying delays. Three numerical examples are given to illustrate the effectiveness of the proposed criteria.     

Passivity of memristor-based BAM neural networks with different memductance and uncertain delays

This paper addresses the passivity problem for a class of memristor-based bidirectional associate memory (BAM) neural networks with uncertain time-varying delays. In particular, the proposed memristive BAM neural networks is formulated with two different types of memductance functions. By constructing proper Lyapunov–Krasovskii functional and using differential inclusions theory, a new set of sufficient condition is obtained in terms of linear matrix inequalities which guarantee the passivity criteria for the considered neural networks. Finally, two numerical examples are given to illustrate the effectiveness of the proposed theoretical results.     

带脉冲的BAM神经网络的全局指数稳定性

利用M矩阵理论,推广的微分不等式和Lyapunov函数,研究了一类带时滞和脉冲的BAM神经网络平衡点的存在唯一性和全局指数稳定性条件.文中推广了以往文献脉冲函数的形式,无需时滞的可导性要求,从而减弱了以往结论的条件,并且可以估计网络的指数收敛速率.     

Global and robust stability of interval Hopfield neural networks with time-varying delays

In this paper, we investigate the problem of global and robust stability of a class of interval Hopfield neural networks that have time-varying delays. Some criteria for the global and robust stability of such networks are derived, by means of constructing suitable Lyapunov functionals for the networks. As a by-product, for the conventional Hopfield neural networks with time-varying delays, we also obtain some new criteria for their global and asymptotic stability.     

变系数变时滞BAM神经网络概周期解的存在性与全局吸引性

利用指数二分性、Banach不动点定理与微分不等式分析技巧,在不要求激活函数有界的条件下,给出了变系数变时滞的BAM神经网络概周期解的存在唯一性和全局吸引性的充分条件．所得结果推广和改进了相应文献的结果。对设计BAM神经网络概周期振荡有重要意义．     

Exponential synchronization of memristive Cohen–Grossberg neural networks with mixed delays

This paper concerns the problem of global exponential synchronization for a class of memristor-based Cohen–Grossberg neural networks with time-varying discrete delays and unbounded distributed delays. The drive-response set is discussed. A novel controller is designed such that the response (slave) system can be controlled to synchronize with the drive (master) system. Through a nonlinear transformation, we get an alternative system from the considered memristor-based Cohen–Grossberg neural networks. By investigating the global exponential synchronization of the alternative system, we obtain the corresponding synchronization criteria of the considered memristor-based Cohen–Grossberg neural networks. Moreover, the conditions established in this paper are easy to be verified and improve the conditions derived in most of existing papers concerning stability and synchronization for memristor-based neural networks. Numerical simulations are given to show the effectiveness of the theoretical results.     

分布时滞双向神经网络的周期解的渐近稳定性

利用重合度连续定理和适当的李亚普诺夫泛函,研究了分布时滞双向神经网络xi^.(t)=-ai(t)xi(t) ∑j=1^m pj,(t)∫-∞^t hji(t-s)fj(yj(s))ds Ii(t),i=1,2……,n,yj^.(t)=-bj(t)yj(t) ∑i=1^n qij(t)∫-∞^t kij(t-s)gi(xi(s))ds Jj(t),j=1,2,…,m的周期解的存在性和渐近稳定性。     

具有时滞的双向联想记忆神经网络的全局渐近稳定性

双向联想记忆模型是两层异联想网络,本文讨论了具有轴突信号传输时滞的双向联想记忆神经网络的全局渐近稳定性,得出了保证神经网络平衡点稳定的几个充分条件,所得到的结论对于具有时滞的连续双向联想记忆神经网络的设计和应用都是很有意义的。     

Exponential synchronization of discontinuous neural networks with time-varying mixed delays via state feedback and impulsive control

This paper investigates drive-response synchronization for a class of neural networks with time-varying discrete and distributed delays (mixed delays) as well as discontinuous activations. Strict mathematical proof shows the global existence of Filippov solutions to neural networks with discontinuous activation functions and the mixed delays. State feedback controller and impulsive controller are designed respectively to guarantee global exponential synchronization of the neural networks. By using Lyapunov function and new analysis techniques, several new synchronization criteria are obtained. Moreover, lower bound on the convergence rate is explicitly estimated when state feedback controller is utilized. Results of this paper are new and some existing ones are extended and improved. Finally, numerical simulations are given to verify the effectiveness of the theoretical results.     

具有时滞的广义BAM脉冲神经网络的全局指数稳定性

本文讨论一类具有时滞的广义双向联想记忆（BAM）脉冲神经网络的指数稳定性．利用Banach空间的不动点定理和Lyapunoy泛函,在不假定激励函数有界、单调和可徽的条件下,得到了平衡点的存在唯一性定理及其为全局指数稳定的充分条件．文末通过实例阐释了所获结论的有效性．