Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.     

Causal interactions between the cerebral cortex and the autonomic nervous system

Mental states such as stress and anxiety can cause heart disease.On the other hand,meditation can improve cardiac performance.In this study,the heart rate variability,directed transfer function and corrected conditional entropy were used to investigate the effects of mental tasks on cardiac performance,and the functional coupling between the cerebral cortex and the heart.When subjects tried to decrease their heart rate by volition,the sympathetic nervous system was inhibited and the heart rate decreased.When subjects tried to increase their heart rate by volition,the parasympathetic nervous system was inhibited and the sympathetic nervous system was stimulated,and the heart rate increased.When autonomic nervous system activity was regulated by mental tasks,the information flow from the post-central areas to the pre-central areas of the cerebral cortex increased,and there was greater coupling between the brain and the heart.Use of directed transfer function and corrected conditional entropy techniques enabled analysis of electroencephalographic recordings,and of the information flow causing functional coupling between the brain and the heart.     

Advances in the reintroduction of rare and endangered wild plant species

Human disturbance and climate change have increased the risk of extinction for rare and endangered wild plant species.One effective way to conserve these rare and endangered species is through reintroduction.In this review,we summarize the advances in wild plant reintroduction from five perspectives:the establishment of reintroduction biology as an important tool for biodiversity conservation;the importance of genetic diversity in reintroduction;reintroduction under global climate change;recruitment limitation in reintroduction;and reintroduction and ecological restoration.In addition,we consider the future of plant reintroduction strategies.     

Erratum to: Maize grain concentrations and above-ground shoot acquisition of micronutrients as affected by intercropping with turnip,faba bean,chickpea, and soybean

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Effects of Temperature and pH on Cell Growth of a Flocculating Microalga (Chlorella vulgaris) JSC-7

微藻是可广泛用于健康食品及水产养殖的饵料,同时,微藻细胞内积累的油脂可作为可再生生物燃料,因此微藻的生长和代谢受到广泛关注。温度和pH对微藻的生物量积累有很大影响,考察不同温度和pH条件下微藻细胞的生长有助于寻找最佳的条件进行微藻的培养。自絮凝小球藻JSC-7（Chlorella vulgaris JSC-7）可实现自沉降采收,有利于降低微藻生产成本,优化其生长条件对更好地利用该微藻具有重要意义。考察了温度（22∽40℃）及pH（6.0∽10.0）对其细胞生长、叶绿素含量和油脂产量的影响。在所选取的温度及pH范围内,JSC-7细胞均可生长,显示该藻种可以适应广泛的温度和pH条件。适合细胞生长的温度依次为31℃〉28℃〉35℃〉25℃,pH依次为7.0〉8.0〉6.0。pH 8.0时生物量和油脂的积累量最多,说明该藻株在弱碱条件下更适合生长和产油。当温度为31℃、pH为7.0时,可获得最高的生长量（OD690=0.941）、叶绿素含量（19 mg/L）及油脂产量（39.07%/克干重）。     

一步法提取植物DNA用于大规模RAPD分析

ＲＡＰＤ技术已广泛地用于植物的系统演化、种群多样性、群体遗传学及杂种种子纯度的鉴定等工作中〔１～３〕。本文基于快速节省的原则对碱液法提取植物ＤＮＡ进行一些改进。１材料与方法ＤＮＡ提取方法取１０～２０ｍｇ幼叶 ,加１００μｌ０ ５ｍｏｌ／ＬＮａＯＨ（或附加０ ５ %～１ ５ %巯基乙醇）研磨后 ,取４０μｌ研磨液加入２００μｌ１００ｍｍｏｌ／ＬＴｒｉｓ ＨＣｌｐＨ７ ６缓冲液（或附加０ ５%不溶性聚乙烯吡咯烷酮 ,ＰＶＰ）中 ,８０００×ｇ离心５ｍｉｎ,上清液即可用于ＰＣＲ反应。ＰＣＲ反应体系含１０ｍｍｏｌ／ＬＴｒｉ…     

近交系小鼠微卫星DNA多态性的研究

随机选择位于小鼠不同染色体上的微卫星引物42对,用PCR技术对C3H、C57、BALB/c、DBA、TA2、T739、B615、BACB/c－nu－nu和SCID等9种实验室常用近交系小鼠微卫星DNA多态性进行了研究。结果显示有信息的40对引物中,9种近交系小鼠在各基因座上均出现一条清晰条带,28个基因座表现为多态性。其中D3Mit22、D7Nds1、D11Mit12、D12Nds2、D15Mit17、D16Mit3、D16Mit4基因座表现为显著多态性。T739、B615和TA2的遗传背景相近,其相似系数分别为90％和85％;其次为TA2、SCID和B615,其相似系数分别为80％和82.5％。结果表明所检测的小鼠符合近交要求,筛选出的引物能典型地反映9个近交系小鼠的品系特异性和遗传背景,可用于常规检测小鼠品系来源和遗传背景等。 Abstract：Forty-two microsatellites DNA loci on different chromosomes in nine kinds of inbred strain mice including C3H,C57,BALB/c,DBA,TA2,T739,B615,BALB/c-nu-nu and SCID were investigated by PCR analysis.It showed that all these mice tested display single allelic gene band with forty pairs of informative primers.Twenty-eight loci are polymorphisms,among which the polymorphisms of D3Mit22,D7Nds1,D11Mit12,D12Nds2,D15Mit17,D16Mit3,and D16Mit4 loci are significant.The genetic background of T739 was similarity with that of B615 and TA2 ,the similarity indices were 90％ and 85％ respectively;and that of TA2 was similarity with SCID and B615,the similarity indices were 80％ and 82.5％.These results suggest that these mice tested meet the request of inbred strain.Screened primers showing marked polymorphisms topically reflect the speciality of strains and genetic backgrounds,which could be used in determining the strains origin and genetic background of mice.     

Using a non-radioisotopic, quantitative TRAP-based method detecting telomerase activities in human hepatoma cells

INTRODUCTIONTelomerase is a ribonucleoprotein complex that plays a critical role in telomeremaintenance and cellular immortality. Telomerase has been considered as tumordiagnostic marker and potential target for cancer therapyll, 2]. A sensitive, reliableand quantitative assay is of high interest in this field. The development of a very sensitive Telomeric Repeat Amplification Protocol (TRAP) for measuring telomerajseactivity in cell extracts has been proved to be an important tool for…