Somatic Cell Cloning Research Articles

Phenotypic effects of somatic cell cloning in the mouse.

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long-term effects of somatic cell cloning.

Bovine embryo technologies

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn. Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Display of different modes of transcription by the promoters of an early embryonic gene, Zfp352, in preimplantation embryos and in somatic cells.

We have previously reported a Krupple-like finger protein gene, Zfp352, which is expressed temporarily in two- to eight-cell mouse embryos. The Zfp352 gene is intron-less in the coding region but carries a solitary 4.3-kb intron in the 5'-untranslated region. In this study, we have analyzed the Zfp352 promoter activity in early embryos and in somatic cells. We determined that the major Zfp352 promoter, designated P1 and located upstream of exon 1, is utilized in both early embryos and in somatic cells. A TATA-like box and a transcription initiator element are discernible in the P1 promoter. We uncovered an alternative promoter, designated P2, in the intron. 5'-Rapid amplification of cDNA ends and real-time RT-PCR experiments indicated that the P2 promoter is weak and is probably fortuitous in early embryos. In somatic cells, however, transfection experiments showed that P2 is as active as P1 as a promoter. Furthermore, P2 appears to be composed of two different subdomains used differentially for transcription initiation in embryos and in somatic cells. Our observations may bear relevance in explaining developmental deficiencies associated with somatic cell cloning experiments.

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells.

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.

Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion.

Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly caused by a combination of factors, such as the time course of meiotic cell-cycle progression after ethanol activation, the degree of spindle rotation, and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell-cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.

Implantation and placental development in somatic cell clone recipient cows.

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.

Cloning of pigs from somatic cells and its prospects.

The technology of somatic cell cloning in pigs is valuable for agricultural and therapeutic purposes. This paper will focus on the current methods of cloning pigs, including our successful microinjection#10; of somatic cell nuclei and its application. #10;

Cell cycle synchronization of mouse skin fibroblasts with nocodazole to obtain G2/M phase rich somatic cell population for nuclear transfer procedure

Objective: G0/G1 phase somatic cells are used commonly for nuclear transfer in animal cloning studies. More recently the same cell-cycle stage somatic cells are utilized also in haploidization experiments with limited success. The present study was designed to establish a protocol to synchronize mouse skin fibroblast cells to obtain somatic cells at the G2/M phase of the cell cycle to provide an alternative source for nuclear transfer experiments that can be used for somatic cell cloning and for haploidization experiments to produce artificial gametes in a more efficient way. Design: Nocodazole is known to inhibit cell cycle progression. Mouse skin fibroblasts were synchronized by using different dosages of nocodazole (ND; 0.25 μg/ml-3.0 μg/ml) combined with different treatment durations. Materials/Methods: Ear biopsies of female B6D2F1 mice were cut into pieces of 1–2 mm, and incubated as tissue explants in DMEM culture medium supplemented with 10 % fetal calf serum. Fibroblast monolayers formed around the tissue explants were harvested and used at passages 5 to 10. To synchronize cells at G2/M, cultures at 70% confluency were supplemented with 0.25–3.0 μg/ml nocodazole in DMEM with 10% fetal calf serum for 3–18 hrs. The cells were then re-suspended and fixed in cold ethanol over night. They were stained with 3 μg/ml propidium iodide, and the cell cycle stage in 10,000 events were analyzed by using a FACScan cytometer. Results: The outcome of the experiment is summarized in the table. Tabled 1G1/G0 (2n)S (2n-4n)G2/M (4n)Control group76.65%3.56%19.64%ND (3 μg/ml) for 3 h70.14%3.97%25.72%ND (0.5 μg/ml) for 3 h71.14%3.56%25.28%ND (3 μg/ml) for 12 h59.36%6.41%34.12%ND (0.5 μg/ml) for 12 h56.96%6.33%36.68%ND (0.5 μg/ml) for 15 h55.07%3.43%41.50%ND (0.25 μg/ml) for 18 h40.82%6.41%52.77% Open table in a new tab Conclusions: The cell cycle of mouse skin fibroblasts can be affected by nocodazole treatment. The present study demonstrates that it is possible to obtain G2/M phase rich somatic cells when applying nocodazole treatment at low dosage combined with long exposure that can provide a new alternative source for nuclear transfer studies in cloning and in haploidization experiments. Supported by: None.

Aberrant patterns of X chromosome inactivation in bovine clones.

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

Oct4 distribution and level in mouse clones: consequences for pluripotency

Somatic cell clones often fail at a developmental stage coincident with commencement of differentiation. The transcription factor Oct4 is expressed during cleavage stages and is essential for the differentiation of the blastocyst. Oct4 expression becomes restricted to the inner cell mass and epiblast. After gastrulation Oct4 is active only in germ cells and is silent in somatic cells. Here, Oct4 and an Oct4-GFP transgene were used as markers for which gene reprogramming could be directly related to the developmental potential of somatic cell clones. Cumulus cell clones initiated Oct4 expression at the correct stage but showed an incorrect spatial expression in the majority of blastocysts. The ability of clones to form outgrowths was reduced, and the outgrowths had low or even undetectable levels of Oct4 RNA or GFP. The quality of GFP signals in blastocysts correlated with the ability to generate outgrowths that maintain GFP expression and the frequency of embryonic stem (ES) cell derivation. Abnormal Oct4 expression in clones is either directly or indirectly caused by reprogramming errors and is indicative of a general failure to reset the genetic program. The abnormal Oct4 expression may be associated with aberrant expression of other crucial developmental genes, leading to abnormalities at various embryonic stages. Regardless of other genes, the variations observed in Oct4 levels alone account for the majority of failures currently observed for somatic cell cloning.

Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

Checkpoint for DNA integrity at the first mitosis after oocyte activation.

Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.

Early death of mice cloned from somatic cells.

Here we report that the lifespan of mice cloned from somatic cells is significantly shorter than that of genotype- and sex-matched controls, most likely due to severe pneumonia and hepatic failure. This finding demonstrates the possibility of long-term deleterious effects of somatic-cell cloning, even after normal birth.

Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer.

Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

Production of transgenic blastocyst of sheep by somatic cell cloning

Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P>0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.

A somatic and germline mosaic mutation in MPZ/P(0) mimics recessive inheritance of CMT1B.

To identify the molecular basis of a demyelinating Charcot-Marie-Tooth disease type 1 (CMT1) with presumed autosomal recessive inheritance. CMT1, an inherited motor and sensory neuropathy with low nerve conduction velocities, is caused by dominantly inherited mutations in the genes of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ/P(0)), and early growth response 2 transcription factor (EGR2/Krox-20). Two young sisters born of clinically and electrophysiologically healthy parents had a severe CMT1 neuropathy of presumed autosomal recessive inheritance. The older sister underwent a nerve biopsy. The authors analyzed PMP22, MPZ/P(0), and EGR2/Krox-20 by automated direct nucleotide sequencing. For rapid mutation detection, they determined the restriction-fragment-length polymorphisms for TaqI in the fluorescein-labeled target DNA sequence amplified by PCR. Nerve biopsy disclosed a demyelinating and remyelinating neuropathy with onion bulb formations. Both sisters had a novel heterozygous G308-->A transition of MPZ/P(0) without any mutation of PMP22 or EGR2/Krox-20. The G308-->A transition was a nonconservative mutation that changed a glycine into a glutamate at the amino acid residue 74 in the extracellular domain of the mature MPZ/P(0). None of 50 healthy controls had the mutation. The healthy mother had a low amount of the mutation in blood (congruent with 20%) as well as in skin, buccal epithelium, and hairs (30%). Because the healthy mother carried clones of somatic mutant cells and had transmitted the G308-->A transition to the affected daughters, she also harbored germline mutant cells. In hereditary demyelinating neuropathies, somatic and germline mosaicism of dominant mutations in the myelin protein genes may mimic autosomal recessive inheritance.

Gonosomal Mosaicism Of A Novel Heterozygous Mutation Of P Causes Charcot‐Marie‐Tooth Neuropathy Type 1b With Apparent Autosomal Recessive Inheritance

Charcot‐Marie‐Tooth neuropathy type 1 (CMT1) is the most common inherited demyelinating neuropathy. It has autosomal dominant inheritance associated with heterozygous mutations in the genes coding the peripheral myelin protein 22 (PMP22), the myelin protein zero (P0) and the early growth response 2 (EGR2) transcription factor. More severe, usually sporadic de‐hypomyelinating neuropathies of infancy are classified as Dejerine‐Sottas syndrome (DSS). Since the original description by Dejerine and Sottas of two affected siblings born of unaffected parents, DSS has long been considered to be autosomal recessive, but it is now known that DSS is also caused by heterozygous mutations that originate de novo in the forementioned genes. True autosomal recessive, usually severe, demyelinating neuropathies are rare and associated with still unknown genes. Two sisters born of unaffected parents suffered from a severe de‐remyelinating neuropathy of infancy consistent with CMT1. Both patients carried a novel heterozygous G308→A transition of P0 without any additional mutation of PMP22 and EGR. The mutation is predicted to cause a Gly74→Glu substitution in the extracellular domain of P0 (P0ex). Gly74Glu is pathogenic because: it was absent in healthy controls; it changes a phylogenetically conserved residue; it is functionally non‐conservative; based on the P0ex crystal, it is predicted to be structurally incompatible with the normal β‐sheet conformation of P0ex. The healthy mother carried a minor proportion of the G308→A transition in white blood cells (≅20 %), fibroblasts, buccal smear and hairs (≅30%). We concluded that she is actually a gonosomal mosaic harboring clones of mutant somatic and germline cells. The Gly74→Glu substitution originated early in the embryogenesis of the mother, before the commitment to germ cells.

Cloning of an Endangered Species (Bos gaurus) Using Interspecies Nuclear Transfer

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Production of Male Cloned Mice from Fresh, Cultured, and Cryopreserved Immature Sertoli Cells1

Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.

Birth of mice after nuclear transfer by electrofusion using tail tip cells.

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.