用高代数小鼠胚胎干细胞制作嵌合体小鼠

为了检验胚胎干的全能性,通过用1日龄卵巢移植后,经体外受精得到的囊胚的饲养建立了小鼠的ES系,并在较高的代数下(第41代)制出了5只毛色嵌合体小鼠,但是没有得到性腺嵌合的小鼠。研究结果证实,高代数的ES细胞仍然具有构建嵌合体的能力。     

南召辛夷SRAP遗传多样性分析及指纹图谱的构建

利用毛细管电泳技术，鉴定SRAP(Sequence-related amplified polymorphisms)引物在34个南召辛夷种系中扩增条带的遗传多样性，聚类分析并构建DNA指纹图谱，为南召辛夷的种系鉴定和分子育种提供理论依据。结果表明：(1)5对SRAP引物共扩增出71条带，其中59条多态性的条带(83.1%),每对引物扩增出多态性位点为9～15条，平均为11.8条；(2)选用多态性高、鉴别能力强的2对引物构建了34份南召辛夷种系基于22个位点的DNA指纹图谱；(3)34份南召辛夷的遗传相似系数为0.58～0.97。UPGMA聚类结果显示，34个南召辛夷样品可分为6大类群，且与采摘地无直接关系。     

中药材人参鉴定技术方法的研究与评价

主要探讨现有的人参鉴定传统方法和分子生物学方法在鉴定过程中的准确性和客观性,综述了聚合酶链式反应(Polymerase Chain Reaction,PCR)、随机扩增多态性DNA标记(Randomly Amplified Polymorphic DNA,RAPD)、限制性片段长度多态性分析(Restriction Fragment Length Polymorphism,RFLP)、扩增片段长度多态性分析(Amplified Fragment Length Polymorphism,AFLP)、多位点探针DNA指纹图谱和微卫星标记技术(SSR)等在人参物种鉴定中的应用,并对每种技术进行了相应的评价.     

用大鼠心肌条件培养基建立来源于C57BL/6J小鼠的ES细胞系

报道一种新的建立C57BL/6J小鼠ES细胞系的方法。采用大鼠心肌条件培养基,在不使用饲养层细胞和白血病抑制因子(LIF)的情况下,从C57BL/6J品系小鼠中建成1个ES细胞系即MESPU 41,成系率为1.0%。MESPU 41细胞为XX型,核型正常率高达89%,表现出XX型ES细胞系少有的稳定性。进行体内分化实验时MESPU 41细胞能发生广泛分化形成畸胎瘤。嵌合体制作实验证实MESPU 41细胞具有嵌合能力,能参与胚胎的发育。采用RT-PCR方法,检测出大鼠心肌细胞有LIF mRNA的表达,这可能与其条件培养基保持ES细胞未分化状态并使X染色体稳定有关。同时,还对大鼠心肌细胞进行了永生化的尝试,共得到了4个永生化克隆,这将进一步简化ES细胞建系和培养工作,为进一步研究ES细胞在体外培养过程中的稳定性开创了新的起点。     

基于Cre-LoxP系统的条件性定点敲入人源hRas基因小鼠的建立

目的 构建基于Cre- LoxP系统的条件性定点敲入人源hRas基因(c-Ha-ras)的小鼠,以获得hRas基因在特定组织器官条件性表达的小鼠模型,用于药物的临床前致癌性安全评价及相关机制研究。方法 首先构建hRas打靶载体,电击法转染入小鼠胚胎干细胞(ES细胞),用正负法筛选阳性ES细胞,通过PCR、 Southern鉴定后,将正确重组hRas基因的ES细胞导入C57BL/6 J小鼠囊胚,移入同步发育的受体鼠子宫,妊娠足月出生的嵌合体小鼠与C57BL/6 J小鼠交配获得杂合子hRasfl/+小鼠。再将杂合子hRasfl/+小鼠间交配获得纯合子小鼠hRasfl/fi,然后与全身组织细胞表达Cre重组酶的Tg (EIIa-cre)小鼠进行交配,获得全身细胞表达hRas基因的hRas-EIIa-cre小鼠,并通过荧光定量PCR方法检测不同日龄胚胎期仔鼠的hRas基因表达水平。结果 成功构建了基于Cre- LoxP系统的人源hRas基因条件性定点敲入小鼠模型的载体,筛选得到的一个正确克隆,电转ES细胞后进行Southern blot鉴定,经过初筛和复筛,共获得12个阳性克隆,挑选A11号克隆ES细胞进行囊胚注射,移植了48枚胚胎,出生9只小鼠,其中6只为嵌合鼠,将嵌合率>50%的雄鼠与野生型C57BL /6 J雌鼠进行交配,出生21只后代,其中鉴定有4只hRasfl/+小鼠;hRasfl/+小鼠间交配出生29只小鼠,其中有14只纯合子hRasfl/fi小鼠;hRasfl/fl小鼠与Tg (EIIa-cre)工具鼠交配6次,未有仔鼠出生;跟踪不同发育时期胚胎中hRas基因的表达发现,E10.5～15.5 d胚胎中检测到hRas基因表达。结论 成功建立运用Cre- LoxP系统建立了带有hRas基因敲入的纯合子小鼠hRasfl/fl,未能得到全身细胞高效表达人源hRas基因的hRas-EIIa-cre小鼠,但为进一步利用hRasfl/fl小鼠模型建立其他组织特异性的条件性基因敲入小鼠模型做好技术储备。     

基于Cre/Loxp系统的Ankle2 基因敲除鼠模型的建立

建立Ankle2基因敲除小鼠模型,为研究该基因在小鼠体内所发挥的重要生理功能提供基础. 本实验利用条件性基因敲除技术,进行打靶载体的设计与构建.将LoxP1st插入到Ankle2基因的第二内含子里,在第五内含子里插入FRT-neo-FRT-LoxP 元件,利用限制性内切酶DNA 及Southern blot 筛选出中靶ES 细胞克隆,随后将发生同源重组的ES细胞注射进C57BL/6J小鼠囊胚中,移入受体小鼠子宫,将得到的嵌合体雄鼠与C57BL/6J雌鼠交配获得Ankle2-Floxed 小鼠. 随后将Ankle2-Floxed 小鼠与全身表达FLP 酶的小鼠进行杂交,将打靶载体中的Neo基因去除,获得F1 代小鼠,F1 代小鼠自交并经PCR 鉴定筛选出Ankle2flox/flox小鼠.Ankle2flox/flox小鼠与全身或组织特异性表达Cre酶小鼠进行杂交,得到Ankle2基因杂合敲出小鼠,该小鼠自交,可获得Ankle2基因纯合敲除的小鼠模型. 该模型为深入研究Ankle2 基因在胚胎发育和衰老发生等中的调控功能提供材料和思路.     

小鼠囊胚的不同遗传背景对形成ES细胞集落的影响

使用正常囊胚经过内细胞团增殖后的离散程序，比较了小鼠C57BL／6品系、129品系和C57BL／6与129杂交的囊胚在形成胚胎干细胞（ES细胞）集落上的差异。C57BL／6品系的正常囊胚经培养后只有17．4％的胚胎出现ES集落，细胞生长迅速但极易分化。129品系为41．0％，细胞生长比较缓慢。而杂交鼠胚易于出现ES细胞集落，高达75．0％，有利于ES细胞系的建立。文中讨论了在嵌合体工作中使用这种杂交鼠胚ES细胞的可能性。     

微卫星与生化位点法对BALB/c小鼠遗传检测比较分析

目的 应用微卫星DNA标记检测BALB/c小鼠的遗传质量,并与现行国家标准生化标记法进行比较,探讨其在近交系小鼠遗传监测中的应用,为建立微卫星DNA检测方法奠定基础。方法 采用20个微卫星基因位点和毛细管电泳技术对BALB/c小鼠进行遗传质量检测,同时采用现行国家标准生化标记法进行检测比较分析。结果 各样品20个微卫星基因位点DNA片段大小相同,没有新的等位基因出现;生化标记检测结果亦显示Akp1等14个生化标记基因均为纯合,个体间生化标记表型均一致,根据国家标准符合品系特征。微卫星DNA标记检测结果与生化标记检测结果一致。结论 微卫星DNA标记可准确可靠、方便快捷地检测近交系小鼠遗传质量,本研究所选的20个微卫星基因位点可用于BALB/c小鼠遗传质量监测。     

两个绵羊品种多位点DNA指纹的研究

利用3种限制性酶HindⅢ,HaeⅢ,HinfⅠ和3种微卫星及小卫星DNA探针(GTG)5,(TG)n,M13检测了2个绵羊品种基因组DNA的多样性.结果表明,具有个体特征的DNA指纹获得依赖于酶/探针的结合.HaeⅢ,HinfⅠ和3种探针都可产生丰富的DNA指纹带谱.DNA指纹表现了高度的个体特异性和多样性,这种多样性表现在带纹的数目、强度和带纹的分布区域上.HindⅢ与3种探针的结合都不能产生清晰的DNA指纹带谱.以特定片段长度区域统计了带纹的相似率,在HaeⅢ/(TG)n组合中,黑头绵羊的平均带纹相似率为0.34,欧洲盘羊为0.50.根据带纹相似率,估算了群体平均杂合度为0.83~0.90.用HaeⅢ/(TG)n组合的指纹相似率,估算了两品种间的相似性指数(0.89)和遗传距离(0.29).     

裸鼠LMLM转移癌模型的鉴定

建立了一株裸鼠淋巴道转移的LMLM瘤系,通过体外培养,建立了稳定的、可长期传代的LMLM癌细胞株,经染色体核型、LDH同功酶染色和Southern blotting分析,确定了LMLM癌的小鼠来源,并通过一系列抗体免疫病理鉴定,进一步确定LMLM是上皮组织来源的小鼠自发性肿瘤。裸鼠自发性皮下肿瘤,经体内皮下接种传代,取转移灶瘤块进行原代培养,得到稳定的可传代的细胞株,形态小而圆,大小一致,贴壁生长,每周传两代,群体细胞倍增时间48小时,经数月的体外传代培养,细胞形态及传代特性无明显变异。体外培养的LMLM癌细胞按常规方法制备染色体标本,可见典型的小鼠“V”形染色体,染色体数范围为36—59,平均数为40.76,有52％细胞染色体数为40条。LMLM癌原位灶及腋下淋巴结转移灶的LDH同功酶电泳行为完全相同,证实淋巴结转移癌细胞确实来源于原位肿瘤的转移,以小鼠肾脏的LDH同功酶谱作为对照,LMLM癌以LDHs为主,而与人小细胞肺癌NCI-H_128的比较,二者LDH的电泳行为完全不同,推论LMLM癌和H_128是两种来源无关的癌系。进一步用Soushern blotting方法鉴别LMLM癌的种系来源,人小细胞肺癌H_128在放射自显影中和人DNA探针呈强阳性杂交,并不受小鼠DNA预杂交影响,而LMLM原位灶和转移灶均不能和人DNA探针杂交。用人DNA预杂交,小鼠肺DNA作为探针,LMLM原位灶和转移灶呈强杂交,人H_128不能与之杂交,这从DNA水平证明,LMLM是小鼠来源的肿瘤。用一系列抗T、B淋巴细胞抗体,抗上皮细胞标志物抗体,抗间叶组织标志物抗体及抗神经内分泌标志物抗体进行免疫组化综合比较分析,结果为LMLM与抗淋巴细胞抗体、抗神经内分泌标志物抗体和抗间叶组织标志物抗体均呈阴性反应,而与部分抗上皮细胞标志物抗体EMA,Keratin反应阳性,故可排除淋巴系统肿瘤可能,不支持小细胞肺癌类的神经内分泌肿瘤,而是一种上皮来源的肿瘤。本实验通过染色体核型、LDH同功酶谱分析及Southern blotting杂交都较肯定地鉴别了LMLM癌的种系来源,并相互验证了实验结果的可靠性,并通过免疫组化鉴定提示该癌为上皮组织来源,LMLM癌的建立和鉴定为肿瘤转移机制研究和肿瘤转移的诊断、治疗提供了一个较好的动物肿瘤转移模型。     

Targetted correction of a mutant HPRT gene in mouse embryonic stem cells

Two recent developments suggest a route to predetermined alterations in mammalian germlines. These are, first, the characterization of mouse embryonic stem (ES) cells that can still enter the germline after genetic manipulation in culture and second, the demonstration that homologous recombination between a native target chromosomal gene and exogenous DAN can be used in culture to modify specifically the target locus. We here use gene targetting functionally to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the ES cell line which has previously been isolated and used to produce an HPRT-deficient mouse. This modification of a chosen gene in pluripotent ES cells demonstrates the feasibility of this route to manipulating mammalian genomes in predetermined ways.     

HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells

Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.     

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.     

Generation of germline-competent induced pluripotent stem cells

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.     

Genetic effects of injection of Rous sarcoma virus DNA into polar plasm of early Drosophila melanogaster embryos

Retroviral proviruses and the transposable elements of eukaryotic genomes are structurally similar. The biological significance of eukaryotic transposable elements has not been examined extensively but it is known that, like prokaryotic transposons, these elements can induce mutations in adjacent genes and cause their transposition. It is of interest to determine whether retroviral proviruses have the same mutagenic and gene transposing ability as transposable elements, particularly because the retrovirus genome is assumed to have originated from transposable elements of lower eukaryotes. The transfer of DNA sequences into animal zygotes or embryos by microinjection is a promising experimental approach for eluxidating their functions: when foreign DNAs were introduced into a mouse germ line, mutations were induced and at least in some mice, the mutation was caused by the insertion of a retroviral sequence. We have introduced Rous sarcoma virus (RSV) DNA into a germ line of Drosophila melanogaster, and describe here the resultant genetic effects.