Epitope-Directed Antibody Elicitation by Genetically Encoded Chemical Cross-Linking Reactivity in the Antigen.

No current methods can selectively elicit an antibody response to a specific conformational epitope in a whole antigen in vivo. Here, we incorporated Nε-acryloyl-l-lysine (AcrK) or Nε-crotonyl-l-lysine (Kcr) with cross-linking activities into the specific epitopes of antigens and immunized mice to generate antibodies that can covalently cross-link with the antigens. By taking advantage of antibody clonal selection and evolution in vivo, an orthogonal antibody-antigen cross-linking reaction can be generated. With this mechanism, we developed a new approach for facile elicitation of antibodies binding to specific epitopes of the antigen in vivo. Antibody responses were directed and enriched to the target epitopes on protein antigens or peptide-KLH conjugates after mouse immunization with the AcrK or Kcr-incorporated immunogens. The effect is so prominent that the majority of selected hits bind to the target epitope. Furthermore, the epitope-specific antibodies effectively block IL-1ß from activating its receptor, indicating its potential for the development of protein subunit vaccines.

Cell Transdifferentiation: A Challenging Strategy with Great Potential.

With the discovery and development of somatic cell nuclear transfer, cell fusion, and induced pluripotent stem cells, cell transdifferentiation research has presented unique advantages and stimulated a heated discussion worldwide. Cell transdifferentiation is a phenomenon by which a cell changes its lineage and acquires the phenotype of other cell types when exposed to certain conditions. Indeed, many adult stem cells and differentiated cells were reported to change their phenotype and transform into other lineages. This article reviews the differentiation of stem cells and classification of transdifferentiation, as well as the advantages, challenges, and prospects of cell transdifferentiation. This review discusses new research directions and the main challenges in the use of transdifferentiation in human cells and molecular replacement therapy. Overall, such knowledge is expected to provide a deep understanding of cell fate and regulation, which can change through differentiation, dedifferentiation, and transdifferentiation, with multiple applications.

E3 ubiquitin ligase OsPIE3 destabilises the B-lectin receptor-like kinase PID2 to control blast disease resistance in rice.

Previous studies have reported that PID2, which encodes a B-lectin receptor-like kinase, is a key gene in the resistance of rice to Magnaporthe oryzae strain ZB15. However, the PID2-mediated downstream signalling events remain largely unknown. The U-box E3 ubiquitin ligase OsPIE3 (PID2-interacting E3) was isolated and confirmed to play key roles in PID2-mediated rice blast resistance. Yeast two-hybrid analysis showed that the armadillo repeat region of OsPIE3 is required for its interaction with PID2. Further investigation demonstrated that OsPIE3 can modify the subcellular localisation of PID2, thus promoting its nuclear recruitment from the plasma membrane for protein degradation in the ubiquitin-proteasome system. Site-directed mutagenesis of a conserved cysteine site (C230S) within the U-box domain of OsPIE3 reduces PID2 translocation and ubiquitination. Genetic analysis suggested that OsPIE3 loss-of-function mutants exhibited enhanced resistance to M. oryzae isolate ZB15, whereas mutants with overexpressed OsPIE3 exhibited reduced resistance. Furthermore, the OsPIE3/PID2-double mutant displayed a similar blast phenotype to that of the PID2 single mutant, suggesting that OsPIE3 is a negative regulator and functions along with PID2 in blast disease resistance. Our findings confirm that the E3 ubiquitin ligase OsPIE3 is necessary for PID2-mediated rice blast disease resistance regulation.

A Small Organic Molecular Catalyst with Efficient Electron Accumulation for Near-unity CO2 Photoreduction.

Molecular catalysis is of great interest to CO2 photoreduction. Various transition metal complexes have been developed as efficient molecular catalysts. However, it remains a challenge to catalyze CO2 reduction by a small organic molecular photocatalyst, as the accumulation of multiple electrons in a small organic molecule is normally difficult for CO2 reduction. We report herein a small organic molecular catalyst can be used for selective reduction of CO2 to CO under visible light irradiation. The turnover number (TON) of CO formation is found to be 400±26 with near 100% selectivity in DMF/H2 O medium. UV-Vis absorption spectroscopy, density functional theory (DFT) calculations, and spectroelectrochemical studies demonstrate that the organic molecular catalyst is capable of accumulating electrons through a 2e- reduced product which shows good stability and is responsible for interacting with CO2 . These findings elucidate an accessible way to develop purely organic molecular catalysts for CO2 reduction by strengthening the electron accumulation.

Exploiting consecutive photoinduced electron transfer (ConPET) in CO2 photoreduction.

The consecutive photoinduced electron transfer (ConPET) process of 1,2,3,5-Tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) in CO2 photoreduction to achieve powerful reducing species has been disclosed by activating a bis(terpyridine)ruthenium(II) complex bearing a high overpotential for selective light-driven reduction of CO2 to CO in homogeneous solution.

Noble metal-free bis-tridentate benzimidazole zinc(II) and iron(II) complexes for selective CO2 photoreduction.

Three noble metal-free metal complexes [Fe(Me-bzimpy)2]2+ (Fe1), [Fe(bzimpy)2]2+ (Fe2) and [Zn(Me-bzimpy)2]2+ (Zn1) were synthesized and studied in the visible light-driven CO2 reduction, where ligands bzimpy and Me-bzimpy were 2,6-bis(1-methyl-1H-benzo[d]imidazol-2-yl)pyridine and 2,6-bis(1H-benzo[d]imidazol-2-yl)pyridine, respectively. It was found that Fe1 displayed the best photocatalytic performance with a turnover number (TON) of 878 and high selectivity up to 99.2% towards CO generation in the presence of an organic thermally activated delayed fluorescence (TADF) photosensitizer, which was more than 10 times that of Fe2 (TONCO = 63) and Zn1 (TONCO = 53). This is attributed to the much higher stability of Fe1 upon reduction, as proved by the cyclic voltammograms of the three complexes. These results highlight the cooperation of ligands and metals in molecular metal complexes for CO2 photoreduction.

Coordination-driven discrete metallo-supramolecular assembly for rapid and selective photochemical CO2 reduction in aqueous solution.

A discrete metallo-supramolecular assembly composed of six iron(ii) cations and twelve redox-active terpyridine fragments has been developed for the highly efficient visible-light-driven reduction of CO2 to CO with a TON of 14 956 and 99.6% selectivity in the presence of an organic thermally activated delayed fluorescence (TADF) photosensitizer 4CzIPN in aqueous solution. The photochemical system proceeds rapidly with a turnover frequency (TOF) of 276 min-1. It is demonstrated that the redox-active terpyridine fragments in the assembly are reduced by the photosensitizer which could further act as an electron reservoir for CO2 reduction, resulting in the highly efficient reduction of CO2. This work shows that discrete metallo-supramolecular assemblies could be used for robust photochemical CO2 reduction.

Water-Assisted Highly Efficient Photocatalytic Reduction of CO2 to CO with Noble Metal-Free Bis(terpyridine)iron(II) Complexes and an Organic Photosensitizer.

Photocatalytic CO2 reduction reaction is believed to be a promising approach for CO2 utilization. In this work, a noble metal-free photocatalytic system, composed of bis(terpyridine)iron(II) complexes and an organic thermally activated delayed fluorescence compound, has been developed for selective reduction of CO2 to CO with a maximum turnover number up to 6320, 99.4% selectivity, and turnover frequency of 127 min-1 under visible-light irradiation in dimethylformamide/H2O solution. More than 0.3 mmol CO was generated using 0.05 µmol catalyst after 2 h of light irradiation. The apparent quantum yield was found to be 9.5% at 440 nm (180 mW cm-2). Control experiments and UV-vis-NIR spectroscopy studies further demonstrated that water strongly promoted the photocatalytic cycle and terpyridine ligands rather than Fe(II) were initially reduced during the photocatalytic process.

[Prokaryotic expression and purification of sheep granulocyte colony stimulating factor for granulosa cell culture].

In order to clarify the regulation of granule cell stimulating factor (GCSF) on granulosa cells, we studied the effect of GCSF on proliferation and apoptosis of in vitro cultured granulosa cells for research on GCSF used in sheep reproduction and breeding. Sheep GCSF protein was prokaryotic expressed and purified. Its bio-activity was measured with M-NSF60 cells. The purified GCSF was added in cell culture medium in experiment groups with non-added as control. Alarmarblue was used to measure cell proliferation, and flow cytometry was used to detect cell cycles and apoptosis. Sheep GCSF could be expressed and purified. Cell activity increased with GCSF concentration from 0.06 to 600 ng/mL at 24 h and 48 h. Cell cycles were significantly different between experiment and control groups at 24 h. Cell ratio of S was significantly reduced (P<0.05) and G2/M phase significantly increased (P<0.05). The apoptosis ratio of experiment group was significantly reduced (P<0.05) at 48 h. In conclusion, GCSF could enhance cell proliferation, inhibit apoptosis, and regulate cell cycles on in vitro cultured sheep granulosa cells.

Complete genome of a unicellular parasite (Antonospora locustae) and transcriptional interactions with its host locust.

Microsporidia are a large group of unicellular parasites that infect insects and mammals. The simpler life cycle of microsporidia in insects provides a model system for understanding their evolution and molecular interactions with their hosts. However, no complete genome is available for insect-parasitic microsporidian species. The complete genome of Antonospora locustae, a microsporidian parasite that obligately infects insects, is reported here. The genome size of A. locustae is 3 170 203 nucleotides, composed of 17 chromosomes onto which a total of 1857 annotated genes have been mapped and detailed. A unique feature of the A. locustae genome is the presence of an ultra-low GC region of approximately 25 kb on 16 of the 17 chromosomes, in which the average GC content is only 20 %. Transcription profiling indicated that the ultra-low GC region of the parasite could be associated with differential regulation of host defences in the fat body to promote the parasite's survival and propagation. Phylogenetic gene analysis showed that A. locustae, and the microsporidian family in general, is likely at an evolutionarily transitional position between prokaryotes and eukaryotes, and that it evolved independently. Transcriptomic analysis showed that A. locustae can systematically inhibit the locust phenoloxidase PPO, TCA and glyoxylate cycles, and PPAR pathways to escape melanization, and can activate host energy transfer pathways to support its reproduction in the fat body, which is an insect energy-producing organ. Our study provides a platform and model for studies of the molecular mechanisms of microsporidium-host interactions in an energy-producing organ and for understanding the evolution of microsporidia.

Epitope-directed antibody selection by site-specific photocrosslinking.

Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human interleukin-1ß and complement 5a. In both cases, more than one-third of hits identified bind to the target epitopes, demonstrating the feasibility and versatility of this method.

Expression, location and biological effects of four and a half LIM domain protein 2 (FHL2) on granulosa cells in ovine.

Previous studies have shown that four and a half LIM domain protein 2 (FHL2) plays an essential role in the regulation of follicular development in mammals. Although the FHL2 genes of human and mouse have been well characterized, the expression and location of FHL2 in ovary and the biological functions of FHL2 on granulosa cells (GCs) of ovine are still not clear. In this study, full-length complementary DNA (cDNA) of FHL2 from ovine follicular GCs was amplified by real-time PCR (RT-PCR). The expression and location of FHL2 in ovary and GCs of ovine were studied by immunohistochemistry and immunofluorescence, and the biological effects of FHL2 on the cell proliferation, cell apoptosis, cell cycles and expression level of related genes of ovine GCs were also explored by overexpression or knockdown of FHL2. The results indicated that FHL2 was expressed in ovine follicular GCs and the sequence of the FHL2 cDNA was consistent with that predicted in GenBank, which did not cause an amino acid change. According to the results, FHL2 was expressed in ovine ovary and mainly located in the cytoplasm and nucleus of GCs. In addition, overexpression of FHL2 significantly reduced the cell viability, promoted the cell apoptosis and decreased the percentage of G0/G1 and S phase cells. RT-PCR showed that overexpression of FHL2 significantly increased the mRNA expression level of Bax and decreased the expression of Bcl-2 and the Bcl-2/Bax mRNA ratio compared with the control group. Besides, the knockdown of FHL2 gene in ovine GCs significantly improved the cell viability, suppressed the cell apoptosis, decreased the mRNA expression level of Caspase-3 gene, increased the Bcl-2/Bax mRNA ratio and increased the percentage of S and G2/M phase cells. Our results suggest that FHL2 may play an important role in the biological functions of GCs in ovine.

Organization of the Addax Major Histocompatibility Complex Provides Insights Into Ruminant Evolution.

Ruminants are critical as prey in transferring solar energy fixed by plants into carnivorous species, yet the genetic signature of the driving forces leading to the evolutionary success of the huge number of ruminant species remains largely unknown. Here we report a complete DNA map of the major histocompatibility complex (MHC) of the addax (Addax nasomaculatus) genome by sequencing a total of 47 overlapping BAC clones previously mapped to cover the MHC region. The addax MHC is composed of 3,224,151 nucleotides, harboring a total of 150 coding genes, 50 tRNA genes, and 14 non-coding RNA genes. The organization of addax MHC was found to be highly conserved to those of sheep and cattle, highlighted by a large piece of chromosome inversion that divided the MHC class II into IIa and IIb subregions. It is now highly possible that all of the ruminant species in the family of Bovidae carry the same chromosome inversion in the MHC region, inherited from a common ancestor of ruminants. Phylogenetic analysis indicated that DY, a ruminant-specific gene located at the boundary of the inversion and highly expressed in dendritic cells, was possibly evolved from DQ, with an estimated divergence time ~140 million years ago. Homology modeling showed that the overall predicted structure of addax DY was similar to that of HLA-DQ2. However, the pocket properties of P1, P4, P6, and P9, which were critical for antigen binding in the addax DY, showed certain distinctive features. Structural analysis suggested that the populations of peptide antigens presented by addax DY and HLA-DQ2 were quite diverse, which in theory could serve to promote microbial regulation in the rumen by ruminant species, contributing to enhanced grass utilization ability. In summary, the results of our study helped to enhance our understanding of the MHC evolution and provided additional supportive evidence to our previous hypothesis that an ancient chromosome inversion in the MHC region of the last common ancestor of ruminants may have contributed to the evolutionary success of current ruminants on our planet.

A high-density BAC physical map covering the entire MHC region of addax antelope genome.

BACKGROUND: The mammalian major histocompatibility complex (MHC) harbours clusters of genes associated with the immunological defence of animals against infectious pathogens. At present, no complete MHC physical map is available for any of the wild ruminant species in the world. RESULTS: The high-density physical map is composed of two contigs of 47 overlapping bacterial artificial chromosome (BAC) clones, with an average of 115 Kb for each BAC, covering the entire addax MHC genome. The first contig has 40 overlapping BAC clones covering an approximately 2.9 Mb region of MHC class I, class III, and class IIa, and the second contig has 7 BAC clones covering an approximately 500 Kb genomic region that harbours MHC class IIb. The relative position of each BAC corresponding to the MHC sequence was determined by comparative mapping using PCR screening of the BAC library of 192,000 clones, and the order of BACs was determined by DNA fingerprinting. The overlaps of neighboring BACs were cross-verified by both BAC-end sequencing and co-amplification of identical PCR fragments within the overlapped region, with their identities further confirmed by DNA sequencing. CONCLUSIONS: We report here the successful construction of a high-quality physical map for the addax MHC region using BACs and comparative mapping. The addax MHC physical map we constructed showed one gap of approximately 18 Mb formed by an ancient autosomal inversion that divided the MHC class II into IIa and IIb. The autosomal inversion provides compelling evidence that the MHC organizations in all of the ruminant species are relatively conserved.

Human Papillomavirus 16 oncoprotein E7 retards mitotic progression by blocking Mps1-MAP4 signaling cascade.

Human epithelial cells can be infected by more than 200 types of human papilloma viruses (HPVs), and persistent HPV infections lead to cervical cancer or other deadly cancers. It has been established that mitotic progression is critical for HPV16 infection, but the underlying mechanism remains unknown. Here, we report that oncoprotein E7 of HPV16 but not HPV18 retards mitotic progression in host cell by direct binding to the C terminus of Microtubule-Associated Protein 4 (MAP4). MAP4 is a novel bona fide target of HPV16E7 protein which binds and recruits the latter to spindle microtubule in mitosis. HPV16E7 protein promotes MAP4 stability by inhibiting MAP4 phosphorylation- mediated degradation to increase the stability of microtubule polymerization and cause an extend mitotic progression. We further uncovered that Mps1 is a kinase of MAP4, and E7-MAP4 binding blocks Mps1 phosphorylation of MAP4, thereby interrupting phosphorylation-dependent MAP4 degradation. Mutations of MAP4 at T927ES928E partially abolished E7-binding capacity and rescued mitotic progression in host cells. In conclusion, our study reveals a molecular mechanism by which HPV16E7 perturbs host mitotic progression by interfering Mps1-MAP4 signaling cascade, which results in an extended infection window and may facilitate the persistent HPV16 infection.

SHON expression predicts response and relapse risk of breast cancer patients after anthracycline-based combination chemotherapy or tamoxifen treatment.

BACKGROUND: SHON nuclear expression (SHON-Nuc+) was previously reported to predict clinical outcomes to tamoxifen therapy in ERα+ breast cancer (BC). Herein we determined if SHON expression detected by specific monoclonal antibodies could provide a more accurate prediction and serve as a biomarker for anthracycline-based combination chemotherapy (ACT). METHODS: SHON expression was determined by immunohistochemistry in the Nottingham early-stage-BC cohort (n = 1,650) who, if eligible, received adjuvant tamoxifen; the Nottingham ERα- early-stage-BC (n = 697) patients who received adjuvant ACT; and the Nottingham locally advanced-BC cohort who received pre-operative ACT with/without taxanes (Neo-ACT, n = 120) and if eligible, 5-year adjuvant tamoxifen treatment. Prognostic significance of SHON and its relationship with the clinical outcome of treatments were analysed. RESULTS: As previously reported, SHON-Nuc+ in high risk/ERα+ patients was significantly associated with a 48% death risk reduction after exclusive adjuvant tamoxifen treatment compared with SHON-Nuc- [HR (95% CI) = 0.52 (0.34-0.78), p = 0.002]. Meanwhile, in ERα- patients treated with adjuvant ACT, SHON cytoplasmic expression (SHON-Cyto+) was significantly associated with a 50% death risk reduction compared with SHON-Cyto- [HR (95% CI) = 0.50 (0.34-0.73), p = 0.0003]. Moreover, in patients received Neo-ACT, SHON-Nuc- or SHON-Cyto+ was associated with an increased pathological complete response (pCR) compared with SHON-Nuc+ [21 vs 4%; OR (95% CI) = 5.88 (1.28-27.03), p = 0.012], or SHON-Cyto- [20.5 vs. 4.5%; OR (95% CI) = 5.43 (1.18-25.03), p = 0.017], respectively. After receiving Neo-ACT, patients with SHON-Nuc+ had a significantly lower distant relapse risk compared to those with SHON-Nuc- [HR (95% CI) = 0.41 (0.19-0.87), p = 0.038], whereas SHON-Cyto+ patients had a significantly higher distant relapse risk compared to SHON-Cyto- patients [HR (95% CI) = 4.63 (1.05-20.39), p = 0.043]. Furthermore, multivariate Cox regression analyses revealed that SHON-Cyto+ was independently associated with a higher risk of distant relapse after Neo-ACT and 5-year tamoxifen treatment [HR (95% CI) = 5.08 (1.13-44.52), p = 0.037]. The interaction term between ERα status and SHON-Nuc+ (p = 0.005), and between SHON-Nuc+ and tamoxifen therapy (p = 0.007), were both statistically significant. CONCLUSION: SHON-Nuce+ in tumours predicts response to tamoxifen in ERα+ BC while SHON-Cyto+ predicts response to ACT.

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation.

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported AlocSWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. AlocSWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT-PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

Epigenetic silencing of TET2 and TET3 induces an EMT-like process in melanoma.

Epithelial-Mesenchymal Transition (EMT) is a critical step in the progression of cancer. Malignant melanoma, a cancer developed from pigmented melanocytes, metastasizes through an EMT-like process. Ten-eleven translocation (TET) enzymes, catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5-hmC), are down regulated in melanoma. However, their roles in the progression and the EMT-like process of melanoma are not fully understood. Here we report that DNA methylation induced silencing of TET2 and TET3 are responsible for the EMT-like process and the metastasis of melanoma. TET2 and TET3 are down regulated in the TGF-ß1-induced EMT-like process, and the knocking down of TET2 or TET3 induced this EMT-like process. A DNA demethylating agent antagonized the TGF-ß-induced suppression of TET2 and TET3. Furthermore, a ChIP analysis indicated that enhanced recruitment of DNMT3A (DNA Methyltransferase 3A) is the mechanism by which TGF-ß induces the silencing of TET2 and TET3. Finally, the overexpression of the TET2 C-terminal sequence partially rescues the TGF-ß1-induced EMT-like process in vitro and inhibits tumor growth and metastasis in vivo. Hence, our data suggest an epigenetic circuitry that mediates the EMT activated by TGF-ß. As an effector, DNMT3A senses the TGF-ß signal and silences TET2 and TET3 promoters to induce the EMT-like process and metastasis in melanoma.

SHON is a novel estrogen-regulated oncogene in mammary carcinoma that predicts patient response to endocrine therapy.

Endocrine therapies are the primary systemic intervention for patients with estrogen receptor-positive (ER(+)) breast cancer. However, a significant proportion of initially responsive ER(+) tumors develop resistance, with relapses occurring in up to 50% of patients. Lack of reliable predictive biomarkers remains an unfilled need for enhanced clinical management of this disease. In this study, we address this need in identifying a novel estrogen-regulated gene called SHON (secreted hominoid-specific oncogene). Enforced expression of SHON in breast cancer cells increased their proliferation, survival, migration, and invasion in vitro. Furthermore, SHON enhanced the oncogenicity of these cells in xenograft models of human breast cancer and was also sufficient to oncogenically transform MCF10A human mammary epithelial cells. Conversely, SHON attenuation mediated by RNA interference- or antibody-based methods reduced the oncogenicity of breast cancer cells. Mechanistic investigations indicated that the oncogenic transforming properties of SHON were mediated by BCL-2 and NF-κB. In primary clinical specimens, SHON was immunohistochemically detected in 62% of breast cancers, in which its expression was positively correlated with ER expression. In this setting, SHON expression predicted a favorable response to endocrine therapy in high-risk patients with ER(+) breast cancer. Taken together, our findings identify SHON as a novel human oncogene with predictive utility in ER(+) breast cancer, perhaps offering a simple biomarker to predict the therapeutic efficacy of antiestrogen therapy in patients with breast cancer.

Handmade cloned transgenic sheep rich in omega-3 Fatty acids.

Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n-3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n-6) into n-3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n  =925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n-3 fatty acid desaturase, accompanied by more than 2-folds reduction of n-6/n-3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation.