HDC细胞与MESPU—13细胞形成嵌合鼠能力的比较研究

ＥＳ细胞嵌合能力的强弱是人们利用ＥＳ细胞获得转基因小鼠时十分关心的问题。本言语通过囊胚显微注射法将１５个左右ＥＳ细胞注入Ｃ５７ＢＬ／６Ｊ品系小鼠３．５天囊胚的囊胚腔中观察嵌合鼠毛色嵌合情况。统计嵌合鼠的出生率;以及用葡萄糖磷酸异构酶（ＧＰＩ）电泳地检测ＥＳ细胞在嵌合鼠体内各种组织和器官的嵌合情况,对于ＨＰＲＴ缺陷（ＨＤＣ）细胞和ＭＥＳＰＵ－１３细胞的嵌合能力我们作了较详细的研究,结果表明ＭＥＳＰＵ     

通过显微注射法构建ES细胞(MESPU 13)嵌合小鼠

为了研究一个新建的小鼠胚胎干细胞系(ES细胞系)MESPU 13,我们将ES细胞显微注射到C57BL/6J小鼠的囊胚中构建嵌合鼠。在注射了2-9个ES细胞的136个囊胚中,127个囊胚(93%)经过3个小时的培养后重新出现囊胚腔。当把119个恢复的囊胚移植到假孕雌鼠的子宫内时,获得63只(52.9%)出生鼠。在59只存活到可以判断毛色阶段的小鼠中,有21只(35.6%)被判定为嵌合鼠。显示了该ES细胞系具有较高的嵌合能力。     

提高利用C57BL/6胚胎干细胞系制作基因打靶小鼠效率的研究

为了提高通过C57BL/6(B6)胚胎干细胞(embryonic stem cells,ES cells)获得基因打靶小鼠的效率,该研究利用经过体外和体内多能性验证的C57BL/6 ES细胞系B6-1-6,开展了37组平行的制作基因打靶小鼠的实验。首先,对不同的囊胚获取的方式进行对比;其次,统计37组实验中的嵌合鼠表观嵌合度与种系遗传效率,并对二者相关性进行分析。对于BALB/c小鼠,自然排卵相较于激素超排能够得到更多囊胚(2.91个囊胚/只vs 0.82个囊胚/只);对于最佳注射个数的探索,利用C57BL/6 ES细胞系B6-1-6进行基因修饰并注射囊胚,注射65~97个胚胎获得阳性打靶动物的可能性为95%;嵌合鼠毛色嵌合率与种系遗传率有一定相关性(r=0.316,P=0.057),而嵌合鼠眼睛颜色与种系遗传效率显著正相关(r=0.328,P0.05)。该研究探索了用于C57BL/6 ES细胞注射的BALB/c囊胚获取方式的优化和最佳的注射数量,以及嵌合鼠表观嵌合度与种系遗传效率相关性等方面,以期为相关的研究提供有益的参考。     

EGFP小鼠ES细胞来源的生殖系嵌合小鼠的获得和鉴定

生殖系嵌合体的获得是实现ES细胞介导的转基因途径的决定步骤,而嵌合体的制作及生殖系嵌合体的获得则是判定ES细胞系是否具有配子分化能力的有效方法。利用一株表达绿色荧光蛋白的杂种ES细胞系制备出嵌合体小鼠,共获得9只表达绿色荧光蛋白的嵌合体小鼠,其中有8只雄性, 1只雌性,目前均发育成健康成年小鼠。流式细胞检测显示了绿色荧光蛋白在嵌合鼠以下器官的表达情况:心(77.96±15.78) %、脾(84.06±3.60) %、肾(42.49±19.79) %、骨髓(52.02±18.78) %。昆明雌鼠与雄性嵌合鼠杂交1代(F1)毛色表型分析显示该株ES细胞具有生殖系嵌合能力。     

不同小鼠品系囊胚受体对C57BL/6胚胎干细胞种系嵌合效率的影响

目的考察不同品系的小鼠囊胚对C57BL/6胚胎干细胞种系嵌合效率的影响,进而提高通过在C57BL/6胚胎干细胞中同源重组制作基因打靶小鼠的效率。方法选取近交系B6(Cg)-Tyrc-2J和BALB/c品系及封闭群ICR作为囊胚供体鼠,通过在TLX3、Ai3K和SL三个不同基因修饰ES细胞的种系嵌合实验中,平行比较三者的囊胚利用率、嵌合鼠获得效率以及种系遗传效率等三个方面,并进一步针对囊胚来源这一因素对效率的影响进行统计学分析。结果三种ES细胞均未能通过B6(Cg)-Tyrc-2J品系囊胚的注射获得种系遗传。而BALB/c品系与ICR品系相比,囊胚利用率明显低于ICR品系(P0.05);嵌合鼠获得效率方面,BALB/c与ICR相当(P=0.115);而种系遗传效率方面,BALB/c品系显著高于其他品系(P0.01)。结论 BALB/c品系在C57BL/6胚胎干细胞种系嵌合效率方面确实具有优势,然而,由于BALB/c囊胚较难获得,并且存在发育延迟和透明带脆性高等问题,而ICR品系在囊胚获得和利用方面的效率明显高于BABL/c,并且也可以支持C57BL/6胚胎干细胞的种系遗传,因此也是C57BL/6胚胎干细胞囊胚注射中较好的选择。     

远交系小鼠胚胎干细胞系的建立及嵌合鼠的获得

ES细胞（Embryonic Stcm Cells)是来源小鼠早期胚胎的多潜能干细胞,它可以在体外大量培养,并以单细胞的形式注射到早期胚胎里,发育为嵌合体,到目前为止,通常使用的129小鼠品系是来源于近交系（inbrcd)小鼠的胚胎。与之相比,远交系小鼠应当具有较强的生命力和抗病能力。曾有人报道过建成了远交系小鼠胚胎干细胞系,但是尚没有见到获得嵌合鼠的报道。有人甚至认为：由于不同品系小鼠所具有的遗传背景不同,有的小鼠不能建成ES细胞系。最近,本实验室在这方面做了有益的探索,成功地建成了远交系小鼠胚胎干细胞系,并在这里报导首例用远交小鼠胚胎干细胞系培育成功嵌合体小鼠。采用源于Swiss小鼠远交群的昆明（KM）品系小鼠囊胚建成了三个小鼠胚胎干细胞系（KE1,KE2,KE5)。核型正常率均达到70％以上。自第八代起分批存。复苏后,培养至第12代,消化成单细胞,通过囊胚显微注射,将其注射到615品系小鼠胚胎。在幸存的幼鼠中获得了一只来源于KE1细胞的嵌合鼠（Table1)。其毛色表现为受体鼠（615）的白色中嵌合有供体鼠（KM)黑褐色（Platc I-A）。嵌合鼠与受体鼠的杂交后代鼠中仍然出现了受体鼠的毛色类型（PlateI-B)。证明：ES细胞能嵌合到生殖腺并形成具有正常功能的配子,从而产生种系嵌合鼠。     

EGFP小鼠ES细胞来源的生殖系嵌合小鼠的获得和鉴定

生殖系嵌合体的获得是实现ES细胞介导的转基因途径的决定步骤，而嵌合体的制作及生殖系嵌合体的获得则是判定ES细胞系是否具有配子分化能力的有效方法。利用一株表达绿色荧光蛋白的杂种ES细胞系制备出嵌合体小鼠，共获得9只表达绿色荧光蛋白的嵌合体小鼠，其中有8只雄性，1只雌性，目前均发育成健康成年小鼠。流式细胞检测显示了绿色荧光蛋白在嵌合鼠以下器官的表达情况：心(77.96±15.78)%、脾(84.06±3.60)%、肾(42.49±19.79)%、骨髓(52.02±18.78)%。昆明雌鼠与雄性嵌合鼠杂交1代（F1）毛色表型分析显示该株ES细胞具有生殖系嵌合能力。     

利用ES细胞建立可诱导的白血病模型

胚胎干细胞（embryonic stem cells,ESCs）是从囊胚的内细胞团分离出来的多潜能干细胞,具有多向分化的能力。将外源基因导入ES细胞建立转基因动物,对于研究外源基因的功能和调控具有一定的价值。载有外源性基因的病毒在感染ES细胞后,可通过囊胚注射获得具有胚系遗传的该转基因动物,并且这一外源基因可以稳定遗传和表达。该研究主要是利用携带hPML-RARα基因的慢病毒感染小鼠ES细胞系（R1）,获得携带该基因的ES细胞,感染后的ES细胞核型正常。在此基础上,将感染后的ES细胞经囊胚注射,获得了携带有hPML-RARα基因的3只嵌合小鼠,其中,有1只具有遗传特性。对嵌合体小鼠与C57杂交的后代给予强力霉素（doxycycline）处理,3天以后骨髓细胞hPML-RARα基因开始表达,这证明了在小鼠体内该外源基因表达的可诱导性。以上证实,已经成功利用ES细胞建立了可诱导的白血病转基因小鼠模型。     

C57BL／6J小鼠ES细胞系的建立及其特性分析

本文报道从C57BL/6J个鼠的囊胚中,建立了三个ES细胞系MESPU17,MESPU18,MESPU19。这些细胞的细胞学特征和强AKP反应,表明这三个细胞系具有干细胞的特征。这三个细胞系均为XY型,正常二倍体核型分别占70％、88％和59％。体外分化可形成简单类胚,体内分化可形成瘤块。与国际上通用的CCE和来自129/ter的ES细胞MESPU13相比,这三个细胞系的ES细胞较大;在体外培养时,生长较慢;细胞也较粘,进行显微注射时,很容易粘在注射针壁上。MESPU17,MESPU18进行了嵌合体制作,以BALB/c和昆明鼠的囊胚为受体,采用囊胚注射法未获嵌合体,但使用昆阴鼠的8细胞胚注射法和共培养法得到嵌合体。     

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.     

大鼠心脏细胞条件培养基对小鼠ES细胞特性的维持

以C19-2和MESPU-13为供试细胞,用克隆测试、传代培养等方法对17种细胞的条件培养基进行了筛选,结果表明,大鼠心脏细胞的条件培养基（RH-CM）具有显著抑制小鼠ES细胞分化、维持其二倍体核型、促进ES细胞贴壁生长的作用。经RH-CM培养10代和20代的小鼠ES细胞在体内外分化能力上仍保留了原ES细胞的多方向分化潜能和特征;RH-CM也可作为小鼠ES细胞培养基的添加物,用含70%RH-CM的ES细胞培养基和小鼠胚胎原代成纤维细胞饲养层（PMEF）培养ES细胞,可长期有效地维持其未分化状态和二倍体核型。RT-PCR检测到大鼠心脏细胞有LIF mRNA表达。     

Studies on Two Factors Affecting the Production of Germline Chimeras by Using HM 1 Cells

ＥＳ细胞系统与基因定位致变相结合,进行基因敲除（ｋｎｏｃｋｏｕｔ）已成为研究基因在生物体内功能的重要手段。在ＥＳ细胞系的建立、外源基因导入ＥＳ细胞、种系嵌合鼠的获得等三个重要环节中,种系嵌合鼠的获得是最关键的一环。由于ＥＳ细胞系统技术复杂、实验条件要求很高,尽管国际上已报导了上百例的基因敲除（ｋｎｏｃｋｏｕｔ）实验,但是到目前为止,我国还无一例在国内条件下获得种系嵌合鼠的正式报道。本研究对影响种系嵌合鼠获得的两种因素（饲养层细胞、受体胚胎种类）进行了比较研究,成功地获得了种系嵌合鼠。将ＨＭ１细胞在ＳＴＯ或ＭＥＦ培养层上培养至２１３３代,注射到不同小鼠的囊胚里,经过恢复培养,移植到假孕的昆明白雌鼠子宫内。由于ＨＭ１细胞来源于粟色的的１２９品系,而胚胎供体鼠的毛色为黑或白色,仔鼠出生一周后即可辨别是否为毛色嵌合鼠。用成年嵌合鼠与其受体胚胎相同品系的小鼠交配,进行种系嵌合鼠鉴定。曾有报导：ＳＴＯ培养层会导致ＥＳ细胞发生核变。我们改用ＭＥＦ培养层,获得嵌合鼠的比率高达４８．６％（Ｔａｂｌｅ１）。不同小鼠胚胎之间存在差异,Ｃ５７ＢＬ／６Ｊ、ＩＣＲ和昆明白三者提供的受体胚胎产生嵌合鼠的比率分别为７１．４％、５５％     

3个不同品系小鼠ES细胞的绿色荧光蛋白标记

用电穿孔法将线性化的质粒pEGFP—N3分别导入来自129／ter、C57BL/6J和BALB／c3个品系的小鼠胚胎干细胞：MESPU—13、MESPU—35和MESPU—62中,经G418筛选、荧光显微镜镜检、阳性克隆扩增、流式细胞仪分选、再扩增以及核型分析等过程,分别得到核型大于85％的被EGFP稳定标记的细胞株5个(129／ter2个、C57BL/6J1个、BALB／c2个),分别命名为MESPU—13／G1和MESPU—13／G2、MESPU—35／G1、MESPU—62／G1和MESPU—62／G2。从不同品系中各选一个增殖生长快、形态典型的标记细胞株,进行碱性磷酸酶染色、oct4群基因产物的表达检测、类胚形成和体内分化鉴定,结果表明所得到的核型正常的、稳定标记的ES细胞系具有原ES细胞的典型特征。     

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electropo-rated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targetin     

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76–80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11–13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells. A. A. Kruglova and E. A. Kizilova contributed equally to this work. This study was financially supported by grants from the Russian Academy of Sciences, Siberian Branch 5.2 and 14.0.     

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.     

小鼠ES细胞种系嵌合体的获得

种系嵌合体的获得是实现ＥＳ细胞介导的转基因途径的决定步骤,ＥＳ细胞种系分化能力的保持是决定种系嵌合的前提条件,而事体的主种系嵌合体的获得则是判定ＥＳ细胞系是否具有种系分化能力的唯一方法,为考察本室新近建立的３种小鼠ＥＳ细胞系ＭＥＳＰＵ２１．ＭＥＳＰＵ２２和ＭＥＳＰＵ２９的种系分化能力,选用近交系Ｃ５７ＢＬ／６Ｊ及远交系ＫＭＷ和ＩＣＲ为受体胚胎提供者,分别通过囊胚注射法和８细胞期桑椹胚注射法进行了嵌     

Developmental potency of teratocarcinoma cells in chimeric embryos

The developmental potential of various teratocarcinomas of different origins was examined by making chimeras with mouse embryos. Of 7 teratocarcinoma lines examined, only 2 were found to contain stem cells having the ability to form live-born chimeras; one was experimentally induced OTTBALB-2 and the other was spontaneously occurring STT-3. The latter showed remarkable ability to colonize mid-gestational fetuses and adults. This result not only demonstrates that EC cells of male primordial germ cells have the ability to form viable chimeras but also suggest that this kind of tumor is a useful source of EC cells to make chimeras. The advantage of using pluripotent cell lines such as EC cells or embryonic stem (ES) cells as a vector for introducing foreign genes into mouse embryos was discussed in relation to the study on gene function and on gene regulation during development.