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。     

从C57BL/6J小鼠胚胎中分离ES细胞系(英文)

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｓ）是来源于小鼠早期胚胎的细胞系,它可以在体外大量培养而不失去其发育的多潜能性。ＥＳ细胞不仅可以用来制作转基因动物,而且能够作为载体进行基因打靶等多种研究。目前,国际上常用的胚胎干细胞系都是来源于１２９小鼠的胚胎。因而,有必要探讨用其它品系小鼠建立ＥＳ系。１９９１年,Ｌｅｄｅｒｍａｎｎ等人首次报道从Ｃ５７ＢＬ／６Ｊ小鼠胚胎中建成了ＥＳ细胞系,但是没有对建立的细胞系进行特性分析。国内柴桂萱等人虽然做了特性分析,但是他们所建细胞系的细胞直径较大,生长速度较慢,不同于常见的ＥＳ细胞系。我们从Ｃ５７ＢＬ／６Ｊ品系小鼠胚胎中共分离了四个ＥＳ细胞系,分别命名为ＣＥ１、ＣＥ２、ＣＥ３、ＣＥ４（Ｆｉｇ．１ａ＆ｂ）。这四个细胞系核型正常率均达到７０％以上。我们检查了ＣＥ２细胞的分化能力,当将ＣＥ２细胞注入同基因型小鼠的皮下后,获得的畸胎瘤（Ｆｉｇ．３）组织切片检查的结果表明：该细胞能够分化成多种组织（Ｆｉｇ．２）。我们也研究了ＥＳ细胞的嵌合能力,用ＩＣＲ小鼠胚胎作为受体胚胎,采用囊胚显微注射法构建嵌合鼠。在幸存的幼鼠中我们获得了来源于ＣＥ２细胞的嵌合鼠（Ｔａｂｌｅ１,Ｆｉｇ．４）。综     

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     

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％     

两个可进入种系的ES细胞系的建立

从１２９／ｔｅｒ小鼠中建立了９个ＥＳ细胞系,它们在核型、生长速度、体内外分化能力等方面显示了各自不同的特点。通过囊胚显微注射法,将ＥＳ细胞注入Ｃ５７ＢＬ／６Ｊ胚胎中,制作了嵌合体,并通过对嵌合体后代毛色的观察,判断了嵌合体生殖细胞的组成。结果表明,ＥＳ细胞系ＭＥＳＰＵ２１、ＭＥＳＰＵ２２都具有很强的种系嵌合能力。比较这两个细胞系与其他细胞系,证明一个好的ＥＳ细胞系必须具备核型正常、生长速度快、体外     

从早期胚胎多能干细胞生成的嵌合鼠

本文利用囊胚注射法将小鼠胚胎多能干细胞－ＣＣＥ细胞注射到发育３天半的昆明和Ｃ３７ＢＬ／６Ｊ小鼠受体囊胚腔内,经假孕鼠借腹怀胎,获３只ＣＣＥ细胞毛色嵌合鼠。实验共注射胚胎６５４个,经培养其恢复成活率７３．８％,胚胎移植后,假母受孕率及产仔率分别为３２．９％和５３％。在所获３只嵌合鼠中,２只为ＣＣＥ－昆明毛色嵌合鼠,１只为ＣＣＥ－Ｃ３７ＢＬ／６Ｊ毛色嵌合鼠,这是国内首次利用胚胎多能干细胞获得嵌合鼠。为     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

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     

源于129S1品系的小鼠胚胎干细胞系的建立及其生物学特性分析

从129S1小鼠早期胚胎的内细胞团分离、培养类胚胎样细胞,经反复传代,成功地建立了129S1小鼠胚胎干细胞系,命名为NM-2细胞系。形态学鉴定具有胚胎干细胞的典型形态特征,正常核型率为80％;呈碱性磷酸酶阳性、表达胚胎干细胞特异性转录因子OCT-4;体内分化后可形成源于三胚层的组织结构;经囊胚腔显微注射后所获得的子代个体中79％具有毛色嵌合表型;雄性嵌合个体中31％发生生殖腺嵌合;同时,通过育种观察到所有生殖腺嵌合体的子代小鼠表型正常。以上结果证实NM-2细胞系为一株具高生殖腺嵌合能力的小鼠胚胎干细胞系。     

pINC－lacZ逆转录病毒载体的构建及其在小鼠胚胎干细胞中的表达

小鼠胚胎干细胞系（ＥＳ）是从囊胚的内细胞团中建立起来的多潜能胚胎干细胞系。ＥＳ细胞系目前正广泛用于将基因打靶后的突变和其它遗传变化导入小鼠的种系中。其中,嵌合鼠的获得是非常必要的一步。这就需要用一个可以广泛表达的基因对ＥＳ细胞进行标记。大肠杆菌β－半乳糖苷酶（β－ｇａｌ）基因的表达在细胞水平就能很容易地观察到,而且对哺乳动物细胞没有任何毒害作用,因而是一个被广泛地用于各种细胞基因表达研究中的报导基因。猿类巨细胞病毒早期（ＳｉＣＭＶＩＥ）启动子是一个广泛用于转基因小鼠研究中的强启动子。然而,该启动子在ＥＳ细胞方面应用的报道尚未见到。本研究用ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,从ｐｓｖ－β－ｇａｌａｃｔｏｓｉｄａｓｅ中得到３．７Ｋｂ的ｌａｃＺ基因片断,将其插入到ｐＩＮＣ载体上（Ｆｉｇ．１）,得到ｐＩＮＣ－ｌａｃＺ（Ｆｉｇ．２）。用ＮｏｔⅠ线性化ｐＩＮＣ－ｌａｃＺ后,电击导入ＭＥＳＰＵ－１３细胞中。ＭＥＳＰＵ－１３为本实验室从１２９／Ｔｅｒ品系小鼠的囊胚中建立的一个ＥＳ细胞系。转化细胞在含有２５０μｇ／ｍｌＧ４１８的培养基上培养两周,进行ＭＥＳＰＵ－１３细胞稳定转化子的筛选。在一次转化实验中,从１ｘ１０７个转化的ＭＥＳ     

品系对小鼠胚胎干细胞分离效率的影响

为了充分利用小鼠胚胎干(ES)细胞,就必须从众多小鼠品系中分离ES细胞系。本研究通过传统的成纤维细胞饲养层法,从CD-1、129/Sv、C57BL/6J和129/Sv×C57BL/6J四种不同遗传背景的小鼠中分离得到12个ES细胞系,而从KM小鼠没有得到ES细胞系。所有的ES细胞系都具有典型的ES细胞特征,AKP染色呈阳性。从四种不同遗传背景的ES细胞系得到了包含多种组织的畸胎瘤;与桑椹胚聚合后,都得到了生殖系嵌合体。结果表明:品系对小鼠ES细胞的分离有显著影响,利用129小鼠以及包含129小鼠遗传背景的杂交小鼠都较容易分离ES细胞,由ES细胞得到生殖系嵌合体的效率在不同品系间有显著差异,从杂交ES细胞比近交ES细胞中更容易得到生殖系嵌合体。     

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.     

Efficacious Production of Viable Germ-Line Chimeras between Embryonic Stem (ES) Cells and 8-Cell Stage Embryos

Many embryonic stem (ES) cell lines have been isolated from various mouse strains, but production of germ-line chimeras has been achieved with only strain 129. This report describes the isolation of a new ES cell line, F1/1, from a mouse blastocyst with the C57BL/6 X CBA male genotype and tests on its ability to produce germ-line chimeras by two techniques, blastocyst injection and 8-cell embryo injection. Chimera production using CD-1 blastocysts as a host was low (20%), as reported by others. But by the 8-cell embryo injection method, in which F1/1 cells were injected into the perivitelline space through a slit in the zona pellucida of 8-cell embryos, chimeric mice with extremely high chimerism were obtained at a rate of 80%. Breeding tests showed that 89% of the fertile males were germ-line chimeras and in most case, the majority of the sperms in their testes were derived from F1/1 cells. This F1/1 cell line with a different genotype from the 129 strain shows high ability to produce functional germ cells, moreover, the 8-cell embryo injection method using F1/1 cells seems to be an efficient way to produce viable germ-line chimeras.     

Survival of porcine inner cell masses in culture and after injection into blastocysts

Isolation of embryonic stem cells has been documented only in the mouse and perhaps the hamster and cow. We report results of experiments designed to determine the effect of age of porcine embryos (6 through 10 d after the first day of estrus) on isolation of cell lines with embryonic stem cell-like morphology. The capacity of fresh and short-term cultured inner cell mass (ICM) cells to differentiate into normal tissues after injection into blastocysts was also measured. Few Day-6 ICM survived in culture to the first passage onto fresh feeder cells, but cell lines with embryonic stem cell-like morphology developed from Day-7 through Day-10 ICM. Isolation of embryonic stem cell-like colonies was achieved at a higher frequency from ICM isolated from older embryos, but embryonic stem cell-like colonies from older embryos also tended to differentiate spontaneously in culture. Viable porcine chimeras were born after injection of fresh ICM into blastocysts that were transferred to recipients for development to term; no chimeras were born from blastocysts injected with ICM subjected to short-term (1 to 6 d) culture. Germ-cell chimerism was confirmed in one of the chimeras. These results document that undifferentiated cells can be removed from porcine blastocysts, transplanted to other embryos, and contribute to development of normal differentiated tissues, including germ cells. Cells with embryonic stem-like morphology can be isolated in culture from ICM at various embryonic ages, but ICM from young blastocysts (e.g., Day-7 embryos) yield embryonic stem cell-like colonies at lower frequency than do ICM from older blastocysts (e.g., Day-10 embryos).     

Establishment of pluripotent cell lines from porcine preimplantation embryos

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.     

Pluripotent Embryonal Stem Cell Lines Can Be Established from Disaggregated Mouse Morulae

Mouse pluripotent embryonal stem ( ES ) cell lines hitherto have been conventionally isolated from the 'inner cell mass' of mouse blastocysts. In this report, I describe a new and simplified method for establishing pluripotent cell lines from mouse morulae of the 16- to 20-cell stage, which were disaggregated by the use of EDTA. From 17 cell lines established in such a way, 7 were characterized with respect to their differentiation potential:
(i) When injected into syngeneic mice, the cells gave rise to solid, fully differentiated teratomas representing derivatives of all three germ layers. (ii) When cultured in suspension in vitro, the cells were able to differentiate into complex organized 'embryoid bodies' analogous to mouse early postimplantation embryos. These results strongly imply that embryonal stem cell lines isolated from mouse morulae are highly homologous to conventionally isolated ES cells.
In addition, my results indicate that murine pluripotent embryonal stem ( ES ) cell lines can be derived with more ease and higher efficiency from disaggregated morulae than from the 'inner cell mass' of blastocysts.     

改进的培养体系在小鼠体细胞核移植及重构胚ES细胞分离培养中的应用

取8周后的雌性昆明小鼠进行超排,取卵母细胞用作核受体,收集卵母细胞周围的卵丘细胞作核供体,进行体细胞核移植。核移植重构胚经SrCl2激活处理6h后,与改良的M16培养液和小鼠输卵管上皮细胞共培养;将发育到早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加含心肌细胞培养液的ES细胞培养液;把孵出的ICM进行消化接种培养,对孵出的ES细胞集落进行鉴定培养。结果显示,以小鼠卵丘细胞为核供体,体细胞核移植重构胚激活率为65．23％,囊胚发育率为11．69％;9个核移植重构囊胚中分离出ES细胞集落,分离率为2．77％;分离出的核移植ES细胞集落具有岛屿状团状隆起结构、碱性磷酸酶染色呈阳性,体外分化可形成类胚体,并能分化成上皮样或梭形细胞。ES细胞集落经常规冻存和复苏后,显示出同冻存前相似的集落形态,并具有较强的增殖能力。实验证实小鼠输卵管上皮细胞、改良的M16培养液及含心肌细胞培养液的ES细胞培养液可以更为成功地运用于小鼠的体细胞核移植及ES细胞的分离培养研究。