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。     

低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型的构建

目的：构建低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型。方法将 Cchl1a3-knock-in打靶载体电转染ES细胞,经过G418和Ganciclovoir筛选阳性ES细胞克隆并用PCR和DNA测序法鉴定。将阳性ES克隆注射到小鼠囊胚,获得嵌合体小鼠。通过杂交获得的杂合子小鼠与FLP小鼠交配繁育获得去neo杂合子小鼠,并用PCR和DNA测序进行鉴定。将去neo杂合子小鼠交配得到纯合子后代,进行生长发育等方面的观察。结果打靶载体成功转染ES细胞,PCR和DNA测序法证实9个ES细胞克隆发生正确的同源重组。通过显微注射获得7只嵌合体小鼠。将嵌合体小鼠交配繁育的杂合子小鼠和FLP小鼠交配获得9只去neo杂合子小鼠,最终得到15只去neo纯合子小鼠。该小鼠在发育至性成熟阶段,精神、饮食及活动状态良好,但是在4个月龄时逐渐出现脱毛,皮肤破溃甚至死亡。结论成功构建Cchl1a3基因 R528H 突变的纯合子小鼠,为研究人类CACNA1S基因功能和阐明低钾型周期性麻痹发生的分子机制奠定了基础。     

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。     

遗传修饰小鼠胚胎干细胞种系嵌合体小鼠的研制

利用显微注射的方法,分别将三株不同类型的经过遗传修饰的中靶ES细胞注射到C57BL/6J小鼠的囊胚中,通过胚胎移植将注射后的囊胚引入受体小鼠子宫中,分别获得了不同整合度的嵌合体小鼠,将高碳合度小鼠与C57BL/6J小鼠杂交,对这些仔鼠进行PCR及Southern鉴定的结果表明,三株修饰后的ES细胞均能整合入生殖系,得到了棕褐色子代鼠,表明获得了种系嵌合体小鼠。     

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。     

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。     

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。     

利用基因诱捕技术进行小鼠基因剔除的初步研究

对利用基因诱捕技术进行小鼠基因剔除做了初步的探索,为进一步应用该技术进行小鼠基因功能研究奠定了基础.利用基因诱捕载体转染小鼠ES细胞,获得了36株neo基因单拷贝整合的诱捕ES细胞,其中14株细胞表达有活性的β半乳糖苷酶.将3株诱捕ES细胞分别经显微注射引入到受体囊胚中,再植入假孕母鼠的子宫中使其发育成小鼠.两株细胞得到了程度不同的嵌合体小鼠,其中一株诱捕ES细胞整合至生殖系.利用质粒拯救实验获得了诱捕载体整合位点附近的基因组序列,通过序列比对发现被诱捕的基因可能是一个新基因.X-gal染色结果显示,该基因的表达局限于小鼠腹部及肢芽的部位.     

遗传背景对小鼠四倍体胚胎发育的影响

不同遗传背景的小鼠2-细胞期胚胎经过电融合后,胚胎的融合效率和四倍体胚胎的发育能力存在着一定的差异。本试验采用C57(C57×C57)、ICR(ICR×ICR)、BALB/c(BALB/c×BALB/c)、B6D2F2(B6D2F1×B6D2F1)、B6C3D2F2(B6C3F1×B6D2F1)品系的二倍体2-细胞期胚胎在相同的条件下经过电融合处理,结果表明:小鼠四倍体胚胎的获得效率受小鼠遗传背景的影响,远交系小鼠胚胎B6D2F2和B6C3D2F2的融合率显著高于近交系C57,ICR和BALB/c(P<0.05);四倍体胚胎在体外的发育情况也受其遗传背景的影响,在桑椹胚发育率和囊胚发育率上B6D2F2和B6C3D2F2品系的四倍体胚胎都显著高于C57和BALB/c品系的四倍体胚胎(P<0.05);杂合和纯系遗传背景的小鼠四倍体胚胎囊胚细胞数目相比具有显著差异(P<0.05或P<0.01);不同遗传背景的小鼠四倍体胚胎着床率间不存在显著差异(P>0.05);杂合背景的小鼠四倍体胚胎得到5只发育至13.5dpc(dayspostcoitum,dpc)的胎儿,纯合背景的小鼠四倍体胚胎得到0只发育至11dpc的胎儿。     

Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells

Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).     

Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice

The completely embryonic stem (ES) cell-derived mice (ES mice) produced by tetraploid embryo complementation provide us with a rapid and powerful approach for functional genome analysis. However, inbred ES cell lines often fail to generate ES mice. The genome of mouse ES cells is extremely unstable during in vitro culture and passage, and the expression of the imprinted genes is most likely to be affected. Whether the ES mice retain or repair the abnormalities of the donor ES cells has still to be determined. Here we report that the inbred ES mice were efficiently produced with the inbred ES cell line (SCR012). The ES fetuses grew more slowly before day 17.5 after mating, but had an excessive growth from day 17.5 to birth. Five imprinted genes examined (H19, Igf2, Igf2r, Peg1, Peg3) were expressed abnormally in ES fetuses. Most remarkably, the expression of H19 was dramatically repressed in the ES fetuses through the embryo developmental stage, and this repression was associated with abnormal biallelic methylation of the H19 upstream region. The altered methylation pattern of H19 was further demonstrated to have arisen in the donor ES cells and persisted on in vivo differentiation to the fetal stage. These results indicate that the ES fetuses did retain the epigenetic alterations in imprinted genes from the donor ES cells.     

At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation

Chimeric or entirely embryonic stem (ES) cell-derived mice ("ES mice") can be produced by injecting ES cells into diploid (2n) or tetraploid (4n) host blastocysts, respectively. Usually, between 10 and 15 ES cells are injected into the host blastocyst, but it is not clear how many of the injected cells contribute to the somatic lineages, thus serve as "founder cells" of the embryo proper. We have used genetically labeled ES cells to retrospectively determine the number of founder ES cells that generate the somatic lineages of chimeric and of ES mice. ES cell clones individually labeled with provirus were mixed in equal numbers and injected into 2n or 4n blastocysts to generate chimeric or ES mice. Southern analysis of DNA from the resulting animals indicated that the somatic lineages were most often derived from one or two and sometimes from up to three founder ES cells. The number of founder cells was independent of the total number of cells injected into the host blastocysts. Our results are consistent with the notion that constraints of the host embryo restrict the number of ES cells that can contribute to a chimeric or an ES mouse.     

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.     

Establishment of a germ-line competent C57BL/6 embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.     

Culturing in vitro produced blastocysts in sequential media promotes ES cell derivation

Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K(+)) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM-CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM-CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM-CBM versus 18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation.     

Completely ES Cell-Derived Mice Produced by Tetraploid Complementation Using Inner Cell Mass (ICM) Deficient Blastocysts

Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.     

Nuclear transfer-mediated rescue of the nuclear genome of nonviable mouse cells frozen without cryoprotectant

Nuclear transfer (NT) provides an opportunity for clonal amplification of a nuclear genome of interest. Here, we report NT-mediated reprogramming with frozen mouse cells that were nonviable because they were frozen at -80 degrees C for up to 342 days without a cryoprotectant. We derived eight embryonic stem (ES) cell lines from cloned blastocysts by conventional NT procedure and five ntES (nuclear transfer embryonic stem) cell lines by a modified NT procedure in which a whole cell instead of a nucleus was injected into an enucleated oocyte. Chromosome analysis revealed that 12 of 13 ntES cell lines have normal karyotypes. On injection of ntES cells into tetraploid blastocysts to generate clonal mice that are nearly completely ntES-cell derived, live pups were obtained; four clonal mice survived until adulthood. On injection of ntES cells into diploid blastocysts, chimeric mice with a high somatic ES cell contribution were generated; germ-line transmission was obtained. Our findings indicate that chromosome stability and genomic integrity can be maintained in mouse somatic cells after freezing without cryoprotection and that NT and ES cell techniques can rescue the genome of these cells.