Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion

Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.     

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.     

Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological,physiological, and neurological characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the 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. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.     

Embryonic stem cells alone are able to support fetal development in the mouse

The developmental potential of embryonic stem (ES) cells versus 3.5 day inner cell mass (ICM) was compared after aggregation with normal diploid embryos and with developmentally compromised tetraploid embryos. ES cells were capable of colonizing somatic tissues in diploid aggregation chimeras but less efficiently than ICMs of the same genotype. When ICM in equilibrium with tetraploid and ES in equilibrium with tetraploid chimeras were made, the newborns were almost all completely ICM- or ES-derived, as judged by GPI isozyme analysis, but tetraploid cells were found in the yolk sac endoderm and trophectoderm lineage. Investigation of ES contribution in 13.5 day ES in equilibrium with tetraploid chimeras by DNA in situ hybridization confirmed the complete tetraploid origin of the placenta (except the fetal blood and blood vessels) and the yolk sac endoderm. However, the yolk sac mesoderm, amnion and fetus contained only ES-derived cells. ES-derived newborns failed to survive after birth, although they had normal birthweight and anatomically they appeared normal. This phenomenon remains unexplained at the moment. The present results prove that ES cells are able to support complete fetal development, resulting in ES-derived newborns, and suggest a useful route for studying the development of genetically manipulated ES cells in all fetal lineages.     

远交系小鼠胚胎干细胞系的建立及嵌合鼠的获得

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｌｓ）是来源于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（ｉｎｂｒｅｄ）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系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。自第八代起分批冻存,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）的黑褐色（ＰｌａｔｅＩ－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（     

A novel method for somatic cell nuclear transfer to mouse embryonic stem cells

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.     

Developmental potential and behavior of tetraploid cells in the mouse embryo

Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.     

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.     

Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2–6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.     

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.     

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％     

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer’s disease

Gene-targeting technology using mouse embryonic stem (ES) cells has become the “gold standard” for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer’s disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409–421, 2003) harboring 3 mutated genes (APP_swe, Tau_P301L, and PS1_M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F₁ mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.     

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.     

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.     

Establishment of the Embryo-derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer.

The ES ceii lines are embryo-derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16-day-old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. Karyotype was, however, nearly tetraploid. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G-banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines.     

Rapid generation of inducible mouse mutants

We have generated an optimized inducible recombination system for conditional gene targeting based on a Cre recombinase–steroid receptor fusion. This configuration allows efficient Cre-mediated recombination in most organs of the mouse upon induction, without detectable background activity. An ES cell line, was established that carries the inducible recombinase and a loxP-flanked lacZ reporter gene. Out of this line, completely ES cell-derived mice were efficiently produced through tetraploid blastocyst complementation, without the requirement of mouse breeding. Our findings provide a new concept allowing the generation of inducible mouse mutants within 6 months, as compared to 14 months using the current protocol.     

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genomewide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)–mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.     

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.