Mammary Gland-specific Expression Vector Research Articles

Identification, purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands.

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19 ± 0.26mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

Expression of functional plant sweet protein thaumatin II in the milk of transgenic mice

Thaumatin is a kind of natural sweet protein which has the characteristics of high sweetness, low calorie, safe and non-toxic, etc. And it has high commercial value in food processing, medicine and other fields.In this study, we employed a sequence that encode thaumatin II, along with goat beta-casein 5′ and 3′ regulatory elements, to construct a mammary gland-specific expression vector. Transgenic mice were generated using pronuclear microinjection, and the expression of thaumatin and its biological activities were assayed in the milk.PCR and southern blot analysis confirmed that the mice harbor thaumatin II transgenes. Thaumatin II retaining their sweetness was expressed in the mammary glands of transgenic mice, as determined by ELISA, western blot and sweetness intensity test.This research describes an initial step in the production of plant protein thaumatin-sweetened milk from large animals such as cow and goat.

Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells

tPA is a thrombolytic agent widely used in clinical settings. While double gene co-integration into organisms can produce synergistic effects and improved expression levels of the target gene, there are few reports detailing the co-integration of the tPA and gGH genes and an increased expression level of tPA. In order to study this, we obtained monoclonal goat mammary epithelial cell lines with tPA/gGH double gene integration and we analyzed the tPA expression level of single and double gene integration cells. We constructed a mammary gland-specific expression vector PCL25/gGH by using the β-casein gene as the regulatory sequence. The tPA and gGH genes were co-transfected into goat mammary epithelial cells by electrotransfection. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were obtained by PCR detection. tPA expression was induced by prolactin and subsequently, the cell induction solution was assayed after 48 hours by ELISA and Western blotting. The results show that a total of 142 resistant monoclonal cells were obtained including 53 tPA monogenic integration cell lines and 34 tPA/gGH double gene integration cell lines. The rate of double gene integration was 23.9% (34/142). A total of 29 cells were detected to be able to express tPA, of which 12 were single-gene-expressing cells and the corresponding expression rate was 22.6% (12/53). There were 17 double-gene- expressing cells with a corresponding expression rate of 50.0% (17/34). The expression level of tPA in single-gene cells was 7.5-52.0 μg/mL, while in double-gene cells was 40-360 μg/mL, which was significantly greater than that in single- gene cells. The goat mammary epithelial cell lines with tPA/gGH gene integration were successfully obtained by electrotransfection, and we proved that the expression level of tPA in the double gene integration cell lines with tPA/gGH gene integration was significantly increased. Our findings lay the foundation for the additional study of highly expressed transgenic goats and other animals with determination of scientific and clinical utility.

Accurately cleavable goat β-lactoglobulin signal peptide efficiently guided translation of a recombinant human plasminogen activator in transgenic rabbit mammary gland.

Poor expression is the key factor hampering the large-scale application of transgenic animal mammary gland bioreactors. A very different approach would be to evaluate the secretion of recombinant proteins into milk in response to a cleavable signal peptide of highly secreted lactoproteins.We previously reported rabbits harboring mammary gland-specific expression vector containing a fusion cDNA (goat β-lactoglobulin (BLG) signal peptide and recombinant human plasminogen activator (rhPA) coding sequences) expressed rhPA in the milk, but we did not realize the signal peptide contributed to the high rhPA concentration and did not mention it at that time. And the molecular structure and biological characteristics still remain unknown. So, rhPA in the milk was purified and characterized in the present study.rhPA was purified from the milk, and the purity of the recovered product was 98% with no loss of biological activity. Analysis of the N-terminal sequence, C-terminal sequence, and the molecular mass of purified rhPA revealed that they matched the theoretical design requirements. The active systemic anaphylaxis (ASA) reactions of the purified rhPA were negative. Taken together, these results indicated that the goat BLG signal peptide can efficiently mediate rhPA secretion into milk and was accurately cleaved off from rhPA by endogenous rabbit signal peptidase.We have reinforced the importance of a rhPA coding region fused to a cleavable heterologous signal peptide from highly secreted goat BLG to improve recombinant protein expression. It is anticipated that these findings will be widely applied to high-yield production of medically important recombinant proteins.

A novel recombinant human plasminogen activator: Efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats.

BackgroundThromboses is a rapidly growing medical problem worldwide. Low-cost, high-scale production of thrombotic drugs is needed to meet the demand. The production of biomolecules in transgenic animals might help address this issue. To our knowledge, the expression of recombinant human plasminogen activator (rhPA) in goat mammary glands has never been reported before.MethodsWe constructed a mammary gland–specific expression vector, BLC14/rhPA, which encodes only the essential K2 fibrin-binding and P domains of wild-type tPA (deletion mutant of tPA lacking the F, E, and K1 domains), along with the goat β-lactoglobulin gene signal peptide-coding sequence. The mammary gland–specific expression vector BLC14/rhPA was transfected into goat fetal fibroblast cells by electroporation. After selection for 3 weeks by G418, stably transfected cell colonies were obtained. PCR analysis results indicated that 24 of the resistant clones were transgenic cell lines; of these, 8 lines were selected as the donor cells. The positive cells were starved for 72 h with DMEM/F12 medium containing 0.5% FBS and were then used as do. Finally, 256 reconstructed oocytes were transferred into 26 recipients, and 7 of them became pregnant (pregnancy rate, 26.9%). Two kids were obtained (BP21 and BP22). PCR analysis confirmed that both were transgenic goats. To analyze the heredity of the rhPA expressed in BP21 F0 and F1 transgenic goats, the F0 transgenic goat BP21 was mated with a normal male goat to generate an F1 transgenic goat. Enucleated metaphase II (MII) oocytes and positive donor cells were used to reconstruct embryos, which were transplanted into the oviducts of the recipients.ResultsWestern blot results showed a specific 39 kDa band. The rhPA expression level in transgenic goat whey was about 78.32 μg/mL by ELISA. Results of ELISA and the in vitro thrombolysis test (FAPA) showed that specific activity of the rhPA in the milk of F0 and F1 transgenic goats was 13.3 times higher than that of the reteplase reference material.ConclusionThus, we demonstrated that BLC14/rhPA was reasonably effective for expression in the mammary glands of transgenic goats, and was stably inherited by the offspring. This study provides the basis for the large-scale production of biological pharmaceuticals in transgenic animals. The expression of biopharmaceuticals by transgenic animals can be used for pharmacological research and bioactive analysis, and transgenic goats were demonstrated to be promising animals for the large-scale production of thrombolytic biopharmaceuticals.

High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

Transgenesis of Tol2-mediated seamlessly constructed BAC mammary gland expression vectors in Mus musculus

Bacterial artificial chromosomes (BACs) are vectors that are capable of carrying gene fragments of up to 300kb in size, and in theory, harbor cis-regulatory elements that are necessary for the expression of specific genes. Therefore, BACs can effectively alleviate or even eliminate the position effect induced by gene-integration, rendering these as ideal expression vectors of exogenous genes. However, the number of relevant studies involving BACs as vectors of exogenous genes are limited. In the present study, we converted the BAC regulatory region of the Mus musculus Wap gene into a mammary gland-specific expression vector. Using the galK-based positive-negative selection method, we seamlessly replaced the Wap gene in a BAC with Homo sapiens GPX3, MT2, and Luc genes while keeping the original mammary gland-specific regulatory sequence intact, without introducing any extra sequences (Loxp/Frt). To improve the efficiency of creating BAC transgenic mice, we used a Tol2 transposon system optimized for mammalian codons and eliminated 100kb of sequence from the BAC 5′ end (173kb), which resulted in an 8.5% rate of successful gene transmission via pronuclear injection. The results of the present study indicate that seamlessly constructed BAC expression vectors can be used for the transmission of the GPX3 gene.

Construction and in vivo evaluation of a mammary gland-specific expression vector for human lysozyme

A mammary gland-specific expression vector p205C3 was constructed with the 5′- and 3′-flanking regions of β-lactoglobulin gene and the first intron of β-casein gene of Chinese dairy goat as regulatory sequences. Human lysozyme (hLYZ) cDNA from mammary gland was cloned into p205C3 and the recombinant vector was used to generate transgenic mice by microinjection. Based on the lysoplate assay, four female offspring of one male founder were detected expressing recombinant hLYZ in their milk at the levels of 5–200 mg/l, and the expressed protein had the same molecular weight as that of normal hLYZ. Besides mammary glands, ectopic expressions were also found in the spleens and the small intestines of the transgenic mice. Among the offspring, the female transgenic mice maintained and expressed the transgene stably with a highest expression level of 750 mg/l. Therefore, p205C3 could be used to develop animal mammary gland bioreactors expressing hLYZ.

Construction of Mammary Gland-specific and Effective Expression Vector for Mammary Gland Bioreactor

Construction of Mammary Gland-specific and Effective Expression Vector for Mammary Gland Bioreactor

Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line.

The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transgenic buffalo for therapeutic use. We have constructed mammary gland specific vector containing human insulin gene and there expression efficiency was checked into in vitro cultured buffalo mammary epithelial cells (BuMECs). Human pro-insulin coding region was isolated from human genomic DNA by intron skipping PCR primer and furin cleavage site was inserted between B-C and C-A chain of human insulin by overlap extension PCR. A mammary gland-specific buffalo beta-lactoglobulin promoter was isolated from buffalo DNA and used for human insulin expression in BuMEC cells. The construct was transfected into BuMECs by lipofection method and positive transgene cell clones were obtained by G418 selection after 3 weeks. Expression of hINS in transfected cells were confirmed by RT-PCR, Immunocytochemistry, Western Blotting and ELISA. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/2.0 × 10(6) cells.

Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer

Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to promote mammogenesis. To this end, we constructed a mammary gland-specific expression vector, pcGH, and demonstrated effective GH expression in goat mammary epithelial cells in vitro by ELISA. Then, to produce transgenic offspring that were capable of stable GH expression in vivo, the linearized pcGH vector was electroporated into goat fetal fibroblasts. Cell colonies that were positive for GH were used as donors for nuclear transfer to enucleated oocytes. A total of 253 morulae or blastocytes developed from the reconstructed embryos were transferred to 56 recipients, resulting in 24 pregnancies at day 35. Finally, six transgenic goats were born. PCR detection confirmed the success of the cloning procedure. To observe the mammogenesis of dairy goats, the GH transgenic goats were mated with a completely healthy buck. In the later pregnancy period, the mammary gland of the GH transgenic goats were extensive than non-transgenic goats. These experiments indicated that the pcGH vector was incorporated into the transgenic goats and affected mammogenesis, which laid a solid foundation for elucidating the impact of GH on mammogenesis and lactation performance.

The development of transgenic mice for the expression of large amounts of human lysozyme in milk

Human lysozyme (hLYZ) has important potential applications as antimicrobial medicine and food additive. To develop a robust expression vector that ensures expression of large amounts of hLYZ in milk, here a 26,267bp chimeric mouse whey acidic protein (mWAP)::hLYZ cassette was constructed and used as a mammary gland-specific expression vector, in which a 3,010bp genomic sequence in the 24,466bp mWAP gene locus was substituted by a 4,811bp genomic sequence of hLYZ, exactly from the start codon to the stop codon. Corresponding transgenic mice were generated, and enzymatically-active hLYZ was expressed at 18.4-35gl(-1) in the milk of most transgenic mouse lines. Our transgenic mice carrying chimeric mWAP::hLYZ represent a model system for cost-effective production of hLYZ.

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

Construction and functional study of pGN, a mammary gland-specific expression plasmid.

The aim of this study was to construct a mammary gland-specific expression vector, pGN, and to validate its function in expressing growth hormone (GH) both in vitro and in vivo. First, the GH gene was amplified and inserted downstream of the b-casein 5'-arm. Next, the neo gene was cloned downstream of the b-casein 3'-arm as a selection marker. To analyze the bioactivity of the pGN plasmid, we expressed pGN in a Bcap-37 cell line and in the goat mammary gland. Quantitative PCR analysis revealed that the expression of GH mRNA in the pGN-transfected group was higher than that of the control group in Bcap-37 cells. Results of a radioimmunoassay and an enzyme-linked immunosorbent assay demonstrated that the pGN-transfected group expressed much more GH protein than the non-transfected group (P < 0.05). Upon injection of the pGN plasmid into the goat mammary gland, GH mRNA and growth hormone receptor mRNA expressions increased 2-fold. In vivo analyses revealed that GH protein expression was higher in the injected group than in the control group. Together, these results strongly demonstrated that the pGN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing GH both in vitro and in vivo.

Scd1 mammary-specific vector constructed and overexpressed in goat fibroblast cells resulting in an increase of palmitoleic acid and oleic acid

Stearoyl-CoA desaturase-1 (Scd1) is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Overexpression of Scd1 in transgenic animals would modify the nutritional value of ruminant-derived foods by increasing the monounsaturated fatty acid (MUFA) and decreasing the saturated fatty acid (SFA) content. The aim of this study was to develop an effective Scd1 vector that is specifically expressed in dairy goat mammary glands. We successfully amplified the goat full length Scd1 cDNA and evaluated its activity in goat ear skin-derived fibroblast cells (GEFCs) by lipid analysis. In addition, we constructed a mammary gland-specific expression vector and confirmed efficient expression of Scd1 in goat mammary epithelial cells (GMECs) by qRT-PCR and Western blot analysis. Fatty acid analysis showed that Scd1-overexpression resulted in an increase in levels of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), from 1.73±0.02% to 2.54±0.02% and from 27.25±0.13% to 30.37±0.04%, respectively (both p<0.01) and the ratio of MUFA to SFA was increased. This work lays a foundation for the generation of Scd1 transgenic goats.

Validation of a recombinant human bactericidal/permeability-increasing protein (hBPI) expression vector using murine mammary gland tumor cells and the early development of hBPI transgenic goat embryos

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland.

Construction of Foot-and-mouth disease virus 2A-based bicistronic expression vector and coexpression of two genes in goat mammary epithelial cells

Using animal mammary glands as bioreactors for producing commercially important proteins is a cutting-edge direction in the field of biotechnology development and application. Dairy goats are an important dairy livestock, with roughage-resistance, fast propagation, long lactation periods and high milk production per bodyweight; these characteristics make dairy goats ideal for use as mammary gland bioreactors. Foot-and-mouth disease virus 2A (FMDV 2A) is an efficient viral cleavage element that mediates proteolytic cleavage independent of the presence of other FMDV sequences. It is often incorporated into recombinant vectors to generate cleavage in the presence of heterologous sequences. To achieve specific co-expression of two heterologous genes in goat mammary gland epithelial (GMGE) cells, a mammary gland-specific bicistronic expression vector, pFIEβ, containing the β-casein 5′ flanking sequence and FMDV 2A, was successfully constructed and the specific expression of human interleukin 2 (hIL-2) and enhanced green fluorescent protein (EGFP) was conducted in primary GMGE cells. Another bicistronic expression vector, pFIEC, driven by the cytomegalovirus promoter, was constructed as a positive control. In cells transfected with pFIEβ and pFIEC, RT-PCR verified the existence of recombinant fusion mRNA of hIL-2 upstream of EGFP within the FMDV 2A cassette fragment and western blot analysis showed the existence of the fusion between hIL-2 and EGFP. It is concluded that FMDV 2A generated specific co-expression of multiple genes for the first time in primary GMGE cells driven by the β-casein promoter.

Construction and function of mammary gland specific goat GH expression vector

The objective of this study was to construct a mammary gland specific gene targeting vector (pLG), which could help the exogenous goat growth hormone (GH) cDNA being integrated into the beta-casein locus of goats and improve the milk production of goat. We cloned the 5' and 3' regulatory sequences of goat's beta-casein gene as homologous arms and inserted them into the Not I/Xho I and Sal I/Cla I multiple clone sites (MCS) of pLoxp II vector respectively, then inserted the GH cDNA into the Xho I MCS of pLoxp II vector and finally got the gene targeting vector pLG. The expression of GH in Bcap-37 cells transfected with plasmid pLG (14.52 ng/mL) was two times higher than that of cells untreated (7.10 ng/mL). While the GH expression of goats injected with plasmid pLG (7.54 ng/mL) was higher than goats untreated or injected with normal saline (6.64 and 6.78 ng/mL). We successfully constructed the mammary gland specific beta-casein gene targeting vector pLG exhibiting bioactivity of transcription and expression of GH both in vitro and in vivo.

The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEO(r) was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase-PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows.

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.