Dimethylated histone H3 lysine 9 is dispensable for the interaction between developmental pluripotency-associated protein 3 (Dppa3) and ten-eleven translocation 3 (Tet3) in somatic cells.

Both developmental pluripotency-associated protein 3 (Dppa3/Stella/PGC7) and dioxygenase ten-eleven translocation 3 (Tet3) are maternal factors that regulate DNA methylation reprogramming during early embryogenesis. In the mouse zygote, dimethylated histone H3 lysine 9 (H3K9me2) attracts Dppa3 to prevent Tet3-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we addressed the interplay between Dppa3 and Tet3 or H3K9me2 in somatic cells. In mouse NIH3T3 cells, the exogenously expressed Dppa3 preferentially accumulated in the cytoplasm and had no effect on Tet3-mediated 5hmC generation. In HeLa cells, the expressed Dppa3 was predominantly localised in the nucleus and could partially suppress Tet3-induced 5hmC accumulation, but this suppressive function was not correlated with H3K9me2. Co-immunoprecipitation assays further revealed an interaction of Dppa3 with Tet3 but not with H3K9me2 in HeLa cells. In cloned zygotes from somatic cells, Dppa3 distribution and 5hmC accumulation in nuclei were not affected by H3K9me2 levels. Taken together, these results suggest that H3K9me2 is not functionally associated with Dppa3 and Tet3 in somatic cells or somatic cell cloned embryos.

Molecular Cloning and Motif Identification of the Sheep Musclin Gene Promoter.

Musclin is a bioactive factor that functions in regulating the muscle growth and metabolism. To investigate the transcriptional regulatory mechanism of the gene, the 1.4 kb musclin promoter in sheep was cloned (GenBank accession: JX966391) and the sequence was analyzed to predict the motifs associated with muscle growth. Next the enhanced green fluorescent protein (EGFP) was selected as the reporter gene and various wild-type and motif-mutant vectors were constructed. The transcriptional regulatory activities were compared by observing the fluorescence strength and detecting the EGFP mRNA expression in C2C12 myoblasts transfected with the vectors. The results showed that the different lengths of promoters could drive the transcription of EGFP and the mutation of some motifs up- or downregulated the activity of the promoter. Furthermore, the electrophoresis mobility shift assay showed that these motifs regulated the musclin gene transcription through binding to the corresponding transcriptional factors in sheep muscle tissue.

Gene Location, Expression, and Function of FNDC5 in Meishan Pigs.

Irisin is a new muscular regulatory factor that is generated by the cleavage of its precursor protein fibronectin type III domain-containing protein 5 (FNDC5). Irisin promotes fat consumption due to its stimulatory role in the browning of the adipocytes in mice. Currently, there is no report on FNDC5 functions in pigs as model animals. In this study, we investigated the expression patterns and functions of FNDC5 in Meishan pigs. Our results showed that FNDC5 gene in Meishan pigs contains five transcripts, all of which can be translated into functional intact irisin proteins. Porcine FNDC5 is mainly expressed in skeletal muscle, with the expression level being significantly higher during the embryonic and juvenile periods than in the adulthood stage. In vitro study showed that FNDC5 stimulates the proliferation and adipogenic differentiation of primary adipocytes isolated from Meishan pigs, and FNDC5 enhances the expression of browning marker genes during adipogenic differentiation. Our study was the first report on FNDC5 expression patterns and functions in pigs. Data from this study provide valuable information related to the study on FNDC5 functions and future development of novel treatment for obesity.

Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs.

Myostatin-deficient mice showed a remarkable hypertrophy of skeletal muscle, with a decreased fat mass and enhanced insulin sensitivity. Currently, it is unclear if the inhibition of myostatin could be used as an approach to treat human obesity and insulin resistance. In this study, we investigated if the inhibition of porcine myostatin has any effect on fat deposition and insulin sensitivity using genetically engineered Meishan pigs containing a myostatin loss-of-function mutation (Mstn -/- ). Our results indicated that, when compared with wild-type pigs, the amount of subcutaneous fat and leaf fat of Mstn -/- pigs were significantly decreased mainly due to the browning of subcutaneous adipose tissue. Additionally, the serum insulin level decreased and the insulin sensitivity increased significantly in Mstn -/- pigs. Moreover, we found a significant increase in levels of insulin receptor and insulin receptor substrate proteins in skeletal muscle of Mstn -/- pigs, which then activating the insulin signaling pathway. Irisin-mediated regulation is not the only pathway for the activation of insulin signal in Mstn -/- skeletal muscle. This study provides valuable insight for the treatment of human obesity and diabetes mellitus.

Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro.

Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression.

Reversible immortalization of sheep fetal fibroblast cells by tetracycline-inducible expression of human telomerase reverse transcriptase.

OBJECTIVES: To achieve reversible immortalization of cells, we design a modified tetracycline-inducible expression (Tet-on) system to conditionally regulate the ectopic expression of human telomerase reverse transcriptase (hTERT) in primary cells. RESULTS: The hTERT gene, hygromycin-resistant gene and all essential elements for achieving tetracycline induction were combined into a single plasmid vector. Sheep fetal fibroblast cells were transfected with the vector and four putative immortalized cell clones were obtained via induction of hTERT expression by doxycycline. These immortalized cells maintained a normal karyotype and showed no transformed phenotype after 250 days continuous culture. When hTERT expression was switched off by withdrawal of doxycycline, the immortalized cells reverted to a normal proliferative state and eventually senesced after limited divisions. CONCLUSIONS: This single-plasmid based Tet-on inducible hTERT expression system can be applied for reversible immortalization of animal cells.

Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN(-/-)) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN(+/+)) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Fel d 1-airway inflammation prevention and treatment by co-immunization vaccine via induction of CD4+CD25-Foxp3+ Treg cells.

Pet allergens are major causes for asthma and allergic rhinitis. Fel d 1 protein, a key pet allergen from domestic cat, can sensitize host and trigger asthma attack. In this study, we report that co-immunization with recombinant Fel d 1 protein (rFel d 1) plus plasmid DNA that contains Fe1 d 1 gene was effective in preventing and treating the natural Fel d 1 (nFel d 1) induced allergic airway inflammation in mice. A population of T regulatory cells (iTreg) exhibiting a CD4+CD25-Foxp3+ phenotype and expressing IL-10 and TGF-ß was induced by this co-immunization strategy. Furthermore, after adoptive transfers of the iTreg cells, mice that were pre-sensitized and challenged with nFel d 1 exhibited less signs of allergic inflammation, AHR and a reduced allergic immune response. These data indicate that co-immunization with DNA and protein mixture vaccine may be an effective treatment for cat allergy.

Dexamethasone-induced skeletal muscle atrophy was associated with upregulation of myostatin promoter activity.

Some recent studies showed that the glucocorticoid-induced muscle atrophy was associated with myostatin, a negative regulator of skeletal muscle. In this study, two experiments were performed to investigate the relationship between the glucocorticoid-induced ultrastructural changes in skeletal muscle and the myostatin gene expression, and to examine in vivo whether the glucocorticoid-induced upregulation of myostatin gene expression is associated with the myostatin promoter activity. In the first experiment, the Kun-Ming mice with similar body weights were treated with high-dose dexamethasone. The results showed that high-dose dexamethasone caused myofibrillar disorganization or degradation and mitochondrial swelling or vacuolization, which were accompanied with the upregulation of myostatin expression. In the second experiment, the mice were treated with the wild-type or GRE (glucocorticoid response elements)-mutant myostatin promoter vector and high-dose dexamethasone alone or together with RU486. The results showed that the mutation of GRE motif resulted in the obvious decrease of the myostatin promoter activity, the high-dose dexamethasone promoted significantly the activity of the wild-type myostatin promoter but did not affect the activity of the GRE-mutant myostatin promoter, and RU486 inhibited the effect of dexamethasone on the wild-type myostatin promoter activity. Taken together, these results suggested that the dexamethasone-induced changes in ultrastructure of skeletal muscle were associated with the upregulation of myostatin gene expression and the upregulation was partly attributed to the binding of glucocorticoid receptor to GRE motifs along myostatin promoter.

Production of transgenic rabbit embryos through intracytoplasmic sperm injection.

The objective of this study was to test if intracytoplasmic sperm injection (ICSI)-mediated gene transfer was an effective method in the production of transgenic rabbit embryos. Rabbit sperm diluted in different media with various pH were treated by freezing without cryoprotectant, and their ability for DNA uptake was determined. In these experiments using production of transgenic rabbit embryos by ICSI, exogenous genes at three concentrations and of two conformation types were used. The rate of DNA association to the sperm seen by rhodamine-tagged DNA encoding green fluorescent protein (GFP) was 90.0%, 92.7%, 91.0%, 91.7%, and 92.3%, respectively in TCM199, DM, DPBS, CZB, and HCZB media. The DNA attachment to sperm was not affected by media pH within the range of 5.4-9.4 (p > 0.05). Expression of GFP first occurred at the 2-cell stage and continued to blastocyst formation. DNA concentration (between 5, 10, and 20 ng/µl) or conformation (linear and circular) had no effect on the production rate of transgenic embryos. These results indicated that genetically modified rabbit blastocysts can be efficiently produced by ICSI technique.

Field evaluation of juvenile in vitro embryo transfer (JIVET) in sheep.

The practicality of using juvenile in vitro embryo transfer (JIVET) on a field scale in China was evaluated in each of three seasons (summer, autumn and winter) from 2006 to 2007. A total of 102 donor Merino lambs (18 summer, 69 autumn and 15 winter) aged 4-8 weeks were stimulated with 4 x 40 mg FSH administered at 12h intervals plus 400 IU PMSG given at the time of the first FSH treatment. Overall, 89.2% (91/102) of the lambs exhibited follicle development and 79.1+/-65.5 (mean+/-S.D.) cumulus-oocyte complexes were recovered per donor lamb. Compared with the groups of summer (84.9+/-55.3) and autumn (83.6+/-70.8) lambs, the number of recovered cumulus-oocyte complexes was significantly decreased in winter (51.4+/-43.7; p<0.05). After recovery, the cumulus-oocyte complexes were matured and fertilized in vitro using frozen-thawed semen and culture in synthetic oviduct fluid medium to the 2-4-c stage of development, when they were transferred surgically in groups of 3-8 (5.33+/-1.47) to the ipsilateral uterine horn of a total of 603 synchronized recipients. The overall mean proportion of cumulus-oocyte complexes developing to 2-c embryos was 61.4% (4308/7013) and differed significantly between seasons (summer 38.5%, autumn 66.1%, winter 74.6%; p<0.01). Pregnancy rate assessed by ultrasound examination approximately 60 days after embryo transfer was 54.4% (328/603) overall, and 36.7% (221/603) of the recipients maintained their pregnancy to full-term, producing an average 1.49 (330/221) offspring, of which 1.21 (267/221) were viable and healthy lambs, per pregnant recipient. Pregnancy rate at day 60 was affected by season (summer 40.5%, autumn 56.7%, winter 55.7%; p<0.05), but did not differ significantly between seasons at full-term (summer 34.2%, autumn 38.9%, winter 30.4%; p>0.05). Based on the number of donors stimulated, the total number of offspring and viable progeny produced per donor lamb in autumn (5.81 and 4.87) was significantly (p<0.01) higher than that of summer (2.79 and 1.94) and winter (4.24 and 3.31). This study showed that each donor lamb after stimulation produced an average of 48.6 transferable embryos that resulted in 4.04 viable and healthy progeny. These results indicate that JIVET is a cost-effective method of multiplying desirable sheep genotypes in China.

Viable rabbits derived from oocytes by intracytoplasmic injection of spermatozoa from an infertile male.

Intracytoplasmic sperm injection (ICSI) is an assisted fertilization technique and has been widely applied in human medicine to overcome some obstacles of infertility. However, this technology has not yet been used as a mainstream technique for animal production, including the rabbit, due to its limited success. The aim of this study was to improve ICSI techniques and establish an efficient ICSI method for rabbits. Spermatozoa used for ICSI were collected from mature New Zealand white male rabbits. They were washed two to three times with HEPES-buffered TCM199 containing 10% FBS and then mixed with 10% polyvinylpyrrolidone (PVP) prior to microinjection. Oocytes were harvested from superovulated donor rabbits after 14-15 h hCG treatment and were fertilized by microinjection of a single living spermatozoon into the ooplasm of each oocyte without additional activation treatment. A total of 317 injected oocytes resulted in the high survival rate of 86.1%. Among the surviving oocytes, 273 were placed into culture dishes for in vitro development. The fertilization, cleavage and blastocyst rates were 59.0%, 88.2% and 45.3% respectively. Furthermore, ICSI embryos were produced with spermatozoa from an infertile male rabbit, and 21 early-stage embryos (2-cell and 4-cell) were surgically transferred into the oviducts of two adult female rabbits. On day 31 after transfer, one out of the two recipients gave birth to two normal and healthy young rabbits. These results demonstrate that rabbit oocytes can be successfully activated and fertilized by the new ICSI protocol. Spermatozoa derived from infertile rabbits can successful fertilize oocytes and produce offspring by the simple ICSI technique.

Roles of extracellular signal-regulated kinase 1/2 on the suppression of myostatin gene expression induced by basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF, FGF-2) has an inhibitory effect on the expression of the myostatin gene in murine C2C12 myoblasts, as shown in our recent investigation. To further verify the regulatory effects of bFGF on the myostatin gene and to better understand its mechanism in skeletal muscle, and to promote clinical applications of bFGF to treat skeletal muscle diseases correlated to muscular dystrophy or AIDS and so on, recombinant human bFGF (rh-bFGF) was added into media and stimulated murine C2C12 myoblasts to investigate the dose-dependent effect of bFGF on suppression of myostatin gene expression and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulatory mechanism. Simultaneously, complete coding sequence of ovine?8 kDa-bFGF gene was inserted into eukaryotic vector pCMV-neo (originated from pEGFP-N1 vector, from which the EGFP gene has been removed), the recombinant plasmid pCMV-neo-bFGF was harvested and injected into the mouse skeletal muscle of posterior limb. Expression levels of bFGF, myostatin, and ERK1/2 genes in murine C2C12 myoblasts and the skeletal muscle were analyzed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. The results showed that bFGF impaired the expression of myostatin gene in a dose-dependent manner in C2C12 cells, with increasing concentration of rh-bFGF, myostatin mRNA declined gradually. In addition, results in skeletal muscle indicated that bFGF also suppressed the expression of the myostatin gene in vivo. Furthermore, we found ERK1/2 participated in the regulatory mechanism of bFGF on the expression of the myostatin gene.

Characterization of the bovine immunoglobulin lambda light chain constant IGLC genes.

To characterize the bovine immunoglobulin lambda light chain constant region (IGLC) genes, we have isolated a bacterial artificial chromosome (BAC) clone by a PCR based approach from a bovine genomic DNA library, constructed using a genital ridge cell line derived from a male Holstein fetus. The positive BAC clone, containing the bovine IGLC genes, was fully sequenced and had a 138 kb insert. Sequence analysis revealed that the bovine immunoglobulin lambda light chain locus consisted of four joining-constant gene recombination units spanning approximately 20 kb DNA in length. A detailed examination of the recombination signal sequences, RNA splicing sites and coding sequences of the four joining-constant gene recombination units suggested that only two IGLC genes (IGLC2 and IGLC3) were functional while the IGLC1 and IGLC4 appeared to be pseudogenes. This conclusion was further confirmed by a series of RT-PCR amplifications, which also showed that among these four genes the IGLC3 was preferentially expressed in cattle. Phylogenetic analysis indicated that the bovine IGLC genes were more closely related to their equivalents in sheep and goats than that to other mammals.

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation.

BACKGROUND: Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation. RESULTS: By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels. CONCLUSION: Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.

Functional analysis of the Myostatin gene promoter in sheep.

Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNPro(W)-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNPro(E(3+5+7)M)-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3-1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin promoter.

Some motifs were important for myostatin transcriptional regulation in sheep (Ovis aries).

Many motifs along the 1.2 kb myostatin promoter (MSTNpro) in sheep have been found by the MatInspecter program in our recent study. To further verify the role of the motifs and better understand the transcriptional regulation mechanism of the myostatin gene in sheep, the reporter gene EGFP (enhanced green fluorescent protein) was selected and the wild-type (W) vector MSTNPro(W)-EGFP or motif-mutational (M) vector MSTNPro(M)-EGFP were constructed. The transcriptional regulation activities were analyzed by detecting the fluorescence strength of EGFP in C2C12 myoblasts transfected with the vectors. The results showed that E-box (E) 3, E4, E5 and E7, particularly E3, E5 and E7, had important effects on the activity of the 1.2 kb sheep myostatin promoter. In addition, we also detected several other important motifs such as MTBF (muscle-specific Mt binding factor), MEF2 (myocyte enhancer factor 2), GRE (glucocorticoid response elements) and PRE (progesterone response elements) along the sheep myostatin promoter by the mutational analysis.

Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilization.

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine (5MeC), the present study detected the DNA methylation patterns of bovine zygotes and preimplantation embryos derived from oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and embryo in vitro culture (IVC). The results showed that: a) paternal-specific demethylation occurred in 61.5% of the examined zygotes, while 34.6% of them showed no demethylation; b) decreased methylation level was observed after the 8-cell stage and persisted through the morula stage, however methylation levels were different between blastomeres within the same embryos; c) at the blastocyst stage, the methylation level was very low in inner cell mass, but high in trophectoderm cells. The present study suggests, at least partly, that IVM/IVF/IVC may have effects on DNA methylation reprogramming of bovine zygotes and early embryos.

Expression of basic fibroblast growth factor results in the decrease of myostatin mRNA in murine C2C12 myoblasts.

During the development and regeneration of skeletal muscle, many growth factors, such as basic fibroblast growth factor (bFGF, FGF-2) and myostatin, have been shown to play regulating roles. bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle, whereas myostatin plays a series of contrasting roles. In order to elucidate whether the expression of bFGF has any relationship with the expression of myostatin in skeletal muscle cells, we constructed a eukaryotic expression vector for the expression of exogenous bFGF in murine C2C12 myoblasts. Quantitative RT-PCR assays indicated that with the increase of the expression of exogenous bFGF gene, the expression of endogenous myostatin gene was suppressed at mRNA level and protein level.

[Transgenic mice produced by intracytoplasmic sperm injection].

In our previous study, normal and fertile mice were successful produced from oocytes following intracytoplasmic sperm injection (ICSI). In the present study, the possibility of producing transgenic embryos and offspring with this procedure was evaluated. After freezing-thawed once using HEPES-CZB medium without cryoprotectants, the cauda sperm from KM fertile male were exposed to the circular or linear pEGFP-N1 DNA for 1 min and then co-injected into metaphase II oocytes of B6D2F1 strain. When the zygotes with two pronuclei were cultured in CZB medium to day 3.5, 39.1% (9/23) of them, derived from oocytes co-injected with sperm head and pEGFP-N1 plasmid DNA, were expressed GFP protein. After transfer of the ICSI embryos with two pronuclei from co-injection of sperm head and foreign DNA, seven recipients delivered 30 pups (23.8%, 30/126). Southern blot results revealed that three of sixteen offspring integrated with GFP and neomycin genes together (18.8 %). Interestingly, all of them were produced from oocytes co-injected sperm head and linear DNA (33.3%, 3/9), while none of seven ICSI offspring integrated either GFP or neomycin gene in the group of co-injection of sperm head and circular plasmid DNA. These results indicated that the high efficiency of transgenic mouse could be produced by ICSI. It may be shown that linear DNA is more easily to integrate into host genome than circular DNA when ICSI was used to produce transgenic animals.