Bovine embryonic stem cells (bESCs) can greatly enhance the understanding of bovine embryonic development and applications for disease-resistance, biomedical, and zoonotic pre-clinical models. However, formative bESCs with distinct morphology and complete differentiation capacity are still unreported. We document here the generation of formative bESCs (bFSCs) which are pluripotent both in vitro and in vivo, and efficiently converted into neural progenitor cells (NPCs) and primordial germ cell-like cells (PGCLCs) by direct differentiation. Transcriptomic analysis reveals these cells exhibited distinct metabolic features from human and mouse ESCs and early embryos. bFSCs contributed to a wide range of cell types within embryonic and extraembryonic tissues after aggregating with mouse and bovine embryos, as confirmed by chimeric experiment and single cell RNA-seq (scRNA-seq). The establishment of bFSCs with dual developmental plasticity represents a milestone for agricultural biotechnology and decoding the underlying mechanism of bona fide bovine pluripotency.

Ruminants play a crucial role in dairy farming, pharmaceuticals, and embryonic stem cell research; thus, it is vital to prevent pregnancy loss and improve reproductive outcomes through a better understanding of placental development. Paternally-expressed gene 10 (PEG10) is a conserved gene essential for placental development in mammals, but its function in ruminants is not well understood. To develop insights into its role in placental development, this study investigated the gene structure and expression of PEG10 in cattle and goats. We found that PEG10's structure was conserved across species, and in the placenta, it retained the ability to bind to its own mRNA. Transcript analysis revealed differential expression patterns of PEG10 at early and late stages of placental development. We identified 70 proteins potentially interacting with PEG10 that were involved in biological processes like metabolism, signal transduction, cell proliferation, and immune responses. These proteins were grouped into seven clusters, associated with pathways such as amino acid degradation, the TCA cycle, longevity regulation, cardiomyopathy, proteasome function, and biosynthesis. Our findings suggest that PEG10 regulates placental development in ruminants by interacting with key proteins like CAST, ITGA6, and FTL, which are responsible for critical cellular processes in placental function.

In mammals, the transcription of transposable elements (TEs) is important for maintaining early embryonic development. Here, we systematically analyzed the expression characteristics of TE-derived transcripts in early embryos by constructing a database of TEs and transcriptome data from goats and using it to study the function of endogenous retroviruses (ERVs) in regulating early embryo development. We found that ERV1 made up the highest proportion of TE sequences and exhibited a stage-specific expression pattern during early embryonic development. Among ERV elements, ERV1 had the potential to encode the Gag protein domain to form virus-like particles (VLPs) in early goat embryos. Knockdown of ERV1_1_574 significantly reduced the embryo development rate and the number of trophoblast cells ( P < 0.05). Transcriptome sequencing analysis of morula embryos showed that ERV1_1_574 mainly regulated the expression of genes related to embryo compaction and trophoblast cell differentiation, such as CX43 and CDX2. In summary, we found that ERV1 expression was essential for early embryonic development in goats through regulation of trophoblast cell differentiation.

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced invitro and invivo developmental rate (P<0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency.

Epigenetic modifications extensively occur in mammalian embryonic development and cell differentiation process. They play an essential role in the reprogramming of nuclei during somatic cell nuclear transfer (SCNT) and subsequent in vitro embryonic development. Recently, SCNT embryos have been verified to contain a subnormal level of histone H3K4 dimethylation (H3K4me2) in contrast to in vitro fertilized embryos. This finding suggested that increasing H3K4me2 levels may ameliorate the aberrant development of cloned embryos. In this study, we investigated the influence of treating donor cells with trans-2-Phenylcyclopropylamine (2-PCPA), a specific inhibitor of lysine-specific demethylase 1 (LSD1), on embryogenesis, H3K4me2 level, and gene expression in cloned goat embryos. Treated goat fetal fibroblast cells (GFFs) with 2-PCPA served as donor cells for subsequent SCNT. Results showed that H3K4me2 levels in treated GFFs increased gradually with the increasing 2-PCPA concentration (p<0.05) and had no obvious influence in cell viability. The 2-PCPA-induced up-regulation of H3K4me2 levels led to G0/G1 cell cycle arrest and the difference was significant at 2M compared with the control group (p<0.05). Interestingly, the development rate of goat SCNT embryos in vitro was significantly improved and aberrant H3K4me2 levels were effectively corrected in 2-PCPA-treated SCNT embryos in contrast to that in SCNT control embryos. Moreover, 2-PCPA treatment promoted the mRNA expression of key developmental genes Oct4 and Sox2 (p<0.05) without affecting the expression levels of imprinted genes IGF2R and H19 in goat SCNT embryos. These results indicated that abnormal H3K4me2 status can be corrected and SCNT embryo development can be promoted through treatment of donor cells with 2-PCPA.Abbreviations: SCNT: somatic cell nuclear transfer; H3K4me2: H3K4 dimethylation; 2-PCPA: trans-2-Phenylcyclopropylamine; LSD1: lysine-specific demethylase 1; GFFs: goat fetal fibroblast cells; IVF: in vitro fertilization; iPS: induced pluripotent stem; PBS: phosphate-buffered saline; IVM: in vitro maturation; RNAPII: RNA polymerase II; HMTs: histone methyltransferase

Quercetin, a plant-derived flavonoid, is frequently used as an antioxidant for efficient anti-oxidative capacity. However, whether quercetin has protective effects on goat sperm and preimplantation embryos against Cd2+-induced oxidative injury is still unclear. So, we researched the influence of quercetin on goat sperm and zygotes respectively under the oxidative stress induced by Cd2+. In our study, quercetin decreased the malonaldehyde (MDA) and reactive oxygen species (ROS) levels caused by Cd2+ in goat sperm (p < 0.05), which facilitated sperm characteristics including motility, survival rates, membrane integrity, and mitochondria activity during storage in vitro and subsequent embryo development (p < 0.05). Moreover, in goat zygotes, quercetin decreased peroxidation products including ROS, MDA, and carbonyl through preserving or maintaining mitochondrial function, gene expression, and anti-oxidative products such as glutathione peroxidase, superoxide dismutase, and catalase, which ameliorated subsequent embryo development and embryo quality (p < 0.05). Taken together, these results suggest that quercetin protects both goat sperm and preimplantation embryos from Cd2+-induced oxidative stress.

Tuberculosis remains a leading health problem worldwide and still accounts for about 1.3 million deaths annually. Expression of the mouse Sp110 nuclear body protein (Sp110) upregulates the apoptotic pathway, which plays an essential role in enhancing host immunity to Mycobacterium tuberculosis (Mtb). However, the mechanism of this upregulation is unclear. Here, we have identified 253 proteins in mouse macrophages that interact with Sp110, of which 251 proteins were previously uncharacterized. The results showed that Sp110 interacts with heat shock protein 5 (Hspa5) to activate endoplasmic reticulum (ER) stress-induced apoptosis, and that this is essential for Sp110 enhanced macrophage resistance to Mtb. Inhibition of the ER stress pathway abolishing the Sp110-enhanced macrophage apoptosis and resulted in increased intracellular survival of Mtb in macrophages overexpressing Sp110 Further studies revealed that Sp110 also interacts with the RNA binding protein, Ncl to promote its degradation. Consequently, the expression of Bcl2, usually stabilized by Ncl, was downregulated in Sp110 overexpressing macrophages. Moreover, overexpression of Sp110 promotes degradation of ribosomal protein Rps3a, resulting in upregulation of the activity of the pro-apoptotic poly (ADP-ribose) polymerase (PARP). In addition, macrophages from transgenic cattle with increased Sp110 expression confirmed that activation of the ER stress response is the main pathway through which Sp110-enhanced macrophages impart resistance to Mtb. This work has revealed the mechanism of Sp110 enhanced macrophage apoptosis in response to Mtb infection, and provides new insights into the study of host-pathogen interactions.

With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated β-lactoglobulin (BLG) gene mono-allelic knockout (BLG +/−) goats and bi-allelic knockout (BLG −/−) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG +/− buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG +/− bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG +/− or BLG −/− bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG +/−, BLG −/− and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6 % for frozen-thawed semen from the BLG +/−, BLG −/− and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG +/− and BLG −/− bucks.

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli, Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1 alpha was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. IL-1 beta was induced by E. coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by E. coli only.GMECs were transfected with IL-1 alpha, IL-1 beta and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacterial challenge. EGFP expression driven by the IL-1 alpha and IL-1 beta promoters was higher in GMECs challenged with E. coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with E. coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland. (C) 2014 Elsevier Ltd. All rights reserved.

Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

SUMMARYSuccessful in vitro maturation (IVM) and oocyte quality both affect the subsequent development of cloned embryos derived from somatic‐cell nuclear transfer (SCNT). Developmental competence is usually lower in oocytes matured in vitro compared with those that matured in vivo, possibly due to insufficient levels of oocyte‐secreted factors (OSFs) and disrupted oocyte–cumulus communication. This study investigated the effects of OSFs secreted by denuded oocytes (DOs) during IVM on the subsequent developmental competence of cloned bovine embryos. Cumulus–oocyte complexes (COCs) from antral follicles of slaughtered‐cow ovaries collected from an abattoir were divided into four groups: COCs co‐cultured with and without DOs in maturation media used for SCNT, as well as COCs co‐cultured with and without DOs in maturation media used for in vitro fertilization (IVF). Based on the developmental competence and embryo quality of bovine embryos generated from these four groups, we found that co‐culturing the COCs with DOs enhanced the in vitro development of IVF and cloned bovine embryos, and potentially generated more high‐quality cloned blastocysts that possessed locus‐specific histone modifications at levels similar to in vitro‐fertilized embryos. These results strongly suggest that co‐culturing COCs with DOs enhances subsequent developmental competence of cloned bovine embryo. Mol. Reprod. Dev. 81: 341–349, 2014. © 2014 Wiley Periodicals, Inc.

Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine β-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus . We report the production of cloned cows carrying human lysozyme gene knock-in β-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

BACKGROUND:PhiC31 integrase is capable of conferring long-term transgene expression in various transfected tissues in vivo. In the present study, we investigated the activity of phiC31 integrase in mouse mammary glands. METHODS:The normal mouse mammary epithelial cell line HC11 was transfected with FuGENE® HD Transfection Reagent (Roche Diagnostics, Shanghai, China). Transfection of the mouse mammary gland in vivo was performed by electrotransfer. Transgene expression was detected by western blotting and an enzyme-linked immunosorbent assay. Genomic integration and integration at mpsL1 was confirmed by a nested polymerase chain reaction. RESULTS:An optimal electrotransfer protocol for the lactating mouse mammary gland was attained through investigation of different voltages and pulse durations. PhiC31 integrase mediated site-specific transgene integration in HC11 cells and the mouse mammary gland. In addition, the site-specific integration occurred efficiently at the ‘hot spot’ mpsL1. Co-delivery of PhiC31 integrase enhanced and prolonged transgene expression in the mouse mammary gland. CONCLUSIONS:The results obtained in the present study show that the use of phiC31 integrase is a feasible and efficient method for high and stable transgene expression in the mouse mammary gland.

Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p < 0.05). The pregnancy rates at 90 and 240 day were significantly lower in groups F2 (4.9% and 3.3%) and F3 (5.4% and 5.4%) compared to groups F1 (23.3% and 16.3%), F4 (25.7% and 18.6%) and F5 (25.9% and 19.8%) (p < 0.05). The cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p < 0.05). Moreover, large offspring syndrome (LOS) incidence in group F5 was significantly lower than those in other groups (p < 0.05). All cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves.

Previous studies have demonstrated that skin stem cells expressing the pluripotency marker stage-specific embryonic antigen 3 (SSEA3) are easier to reprogram into induced pluripotent stem cells (iPSCs) than skin fibroblasts. Furthermore, it is widely speculated that the undifferentiated state may make stem cells more efficient donor cells for somatic cell nuclear transfer (SCNT). In this study, we isolated SSEA3(+) cells from goat skin fibroblast cells (SFCs) using fluorescence-activated cell sorting (FACS) and examined expression of pluripotency markers and in vitro development of cloned embryos following SCNT. Results showed that cell clusters from SSEA3(+) cells were consistently positive for alkaline phosphatase staining and pluripotency markers, Nanog, Oct4, Sox2, and SSEA3. The cleavage rate of cloned embryos derived from SSEA3(+) cells did not differ compared with SFCs (70.5±0.8% and 68.4±2.1%, respectively), but was significantly higher compared with SSEA3(-) cells (64.9±1.6%, p<0.05). The blastocyst rate was significantly increased in the SSEA3(+) cell group compared with the SFC and SSEA3(-) cell groups (30.3±1.2% vs. 21.2±0.9 and 19.0±1.0%, respectively, p<0.05). The quality of cloned blastocysts from SSEA3(+) cells was higher compared with SFCs and SSEA3(-) cells, based on total cell number and number of apoptotic cells per blastocyst. These findings suggest that using SSEA3(+) cells as donors for SCNT is beneficial for enhancing in vitro development and quality of cloned goat embryos.

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

Transgenic animals that express antimicrobial agents in their milk can inhibit bacterial pathogens that cause mastitis. Our objective was to produce human β-defensin-3 (HBD3) transgenic embryos by nuclear transfer using goat mammary epithelial cells (GMECs) as donor cells. Three GMEC lines (GMEC1, GMEC2, and GMEC3) were transfected with a HBD3 mammary-specific expression vector by electroporation. There was a difference (P < 0.05) in the rate of geneticin-resistant colony formation among cell lines GMEC1, GMEC2, and GMEC3 (39 and 47 vs. 19 colonies per 3 × 10(6) cells, respectively). After inducing expression, the mRNA and protein of HBD3 were detected by reverse transcription polymerase chain reaction and Western blot analysis in transgenic cells. Transgenic clonal cells expressing HBD3 were used as donor cells to investigate development of cloned embryos. There were no significant differences in rates of cleavage or blastocyst formation of cloned embryos from transgenic (GMEC1T2 and GMEC2T3) and nontransgenic (GMEC1 and GMEC2) GMECs (72.3 ± 5.0%, 69.5 ± 2.3%, 61.8 ± 4.8%, and 70.0 ± 2%; and 16.8 ± 0.5%, 17.5 ± 0.7%, 16.7 ± 0.9%, and 17.5 ± 0.6%, respectively). However, the fusion rate, cleavage rate, and blastocyst formation rate of cloned embryos from a transgenic clonal cell line (GMEC2T6, 50.7 ± 2.1%, 55.5 ± 2.0%, and 11.1 ± 0.6%) were lower than those of other groups (P < 0.05). We concluded that genetic modification of GMECs might not influence the in vitro development of cloned embryos, but that some of the transgenic clonal cells were not suitable for nuclear transfer to produce transgenic goats, because of low developmental rates. However, transgenic GMECs expressing HBD3 might be used as donor cells for producing transgenic goats that express increased concentrations of β-defensins in their milk.

Achieving high expression levels of recombinant human serum albumin (HSA) for purification is a solution for the large amount of plasma-derived HSA needed in therapeutic applications. Here, we employed phiC31 integrase system and chicken hypersensitive site-4 (cHS4) insulators to construct a HSA expression vector for high-level HSA expression. The phiC31 integrase system mediated efficient transgene integration in bovine mammary epithelial cells (bMECs). A preferred pseudo attP site, which had 38 % identity with the 39 bp wild-type attP sequence, was detected in six out of 55 bMEC colonies. Addition of the cHS4 insulator to the phiC31 integrase system resulted in 8-20-fold increases of HSA expression compared with that of using integrase alone. Moreover, the reverse-oriented cHS4 insulator in the phiC31 integrase system provided the optimal level of HSA expression in bMECs.