[Application of intermittent flap opening in L-shaped surgical incision of calcaneal fracture].

OBJECTIVE: To explore clinical effect of intermittent flap opening technique in L-shaped incision of calcaneal fracture. METHODS: From January 2017 to January 2019, 48 patients with Sanders typeⅡ to Ⅳ calcaneal fractures were treated by open reduction and internal fixation. According to different flap opening techniques, the patients were divided into control group and observation group, 24 patients in each group. In observation group, there were 17 males and 7 females, aged from 20 to 60 years old with an average of(45.12±9.56) years old;7 patients were typeⅡ, 10 patients were type Ⅲ and 7 patients were type Ⅳ according to Sanders classification;3 patients were C0, 16 patients were C1 and 5 patients were C2 according to Tscherne-Gotzen soft-tissue assessment;treated with intermittent flap technique. In control group, there were 19 males and 5 females aged from 20 to 60 years old with an average of (47.32±10.67) years old;7 patients were typeⅡ, 11 patients were type Ⅲ and 6 patients were type Ⅳ according to Sanders classification;2 patients were C0, 18 patients were C1 and 4 patients were C2 according to Tschemc-Gotzen soft-tissue assessment;treated with static flap opening technique. Operation time, flap retraction time, changes of Böhler angle and Gissane angle before and after operation at 3 days, and occurrence of incision complications were observed and compared between two groups. RESULTS: All patients were followed up from 3 to 6 months with an average of(4.52±1.01) months. There were no significant differences in operation time, changes of Böhler angle and Gissane angle before and after operation at 3 days between the two groups(P>0.05);there was statistical difference in flap retraction time between two groups(P<0.05). Occurrence of incision complications in observation group was significantly lower than that in control group (P<0.05). CONCLUSION: Intermittent flap opening technique is superior to static opening technique in reducing incision complications of lateral "L" approach of calcaneus. Single Kirschner wire opening does not affect the exposure, reduction and fixation of fracture during operation.

[Selective U-shaped osteotomy of lateral tibial condyle for the treatment of collapse and comminuted fracture of lateral tibial plateau].

OBJECTIVE: To explore methods and clinical effects of selective U-shaped osteotomy of lateral tibial condyle in treating collapse and comminuted fracture of lateral tibial plateau. METHODS: From January 2014 to October 2019, 15 patients with collapse and comminuted fracture of lateral tibial plateau were treated by selective U-shaped osteotomy of lateral tibial condyle, including 9 males and 6 females. The age of patients ranged from 25 to 70 years old, with an average age of (38.5±7.7) years old. According to ABC classification of condyle fracture of tibial plateau lateral, there were 2 cases of type A, 6 cases of type B, 4 cases of type BC and 3 cases of type C. Five patients were combined with medial plateau fracture, 8 patients were combined with left knee fracture and 7 patients of right knee fracture. The time of treatment after injury ranged from 1 day to 14 days with an average of (3.4±1.2) days. CT of all patients showed that lateral tibial plateau collapsed more than 2 mm, more than 2 pieces of bones were crushed and broken, and lateral tibial condyle cortex was intact. At follow-up of 12 months after operation, Rasmussen's anatomical grading system was used to evaluate fracture reduction. Rasmussen's functional grading system were used to evaluate knee joint function. RESULTS: Selective U-shaped osteotomy was successfully complated in 15 patients at one time, and operation time ranged from 55 to 110 min, with an average time of (85.6±20.0) min. The lateral plateau operation ranged from 20 to 60 min with an average time of(30.5±10.5) min. All patients were followed up for 12 to 24 months with an average of (14.6±2.5) months. Fracture healing time was 12 to 24 weeks, with an average of (13.6±3.6) weeks. At follow-up 12 months after operation, by Rasmussen's grading system, anatomical score of knee joint ranged from 14 to 18 points, with an average score of (17.5±0.3) points, of which 13 cases were excellent and 2 cases were good. The functional score ranged from 13 points to 30 points, with an average score of (26.8±2.5) points. Among them, 12 cases were excellent, 1 case was good, 2 cases were fair. Two patients suffered 2 mm and 4 mm loss of lateral tibial plateau, 1 case of knee joint 5 ° valgus, 1 case of stiff joints (10 ° to 100 °). No common peroneal nerve injury, important vascular injury, postoperative infection, internal fixation failure and other serious complications was found. CONCLUSION: The use of selective lateral tibial condyle "U"- shaped osteotomy approach is an effective and reliable method to treat the collapse and comminuted fracture of the lateral tibial plateau. It has the advantages of simple surgical incision, direct fracture exposure, accurate repositioning and fixation, short operation time and few complications.

[EFFECTIVENESS OF OPEN REDUCTION AND INTERNAL FIXATION WITH LOCKING ANATOMICAL PLATE FOR TREATING VALGUS IMPACTED PROXIMAL HUMERUS FRACTURE].

OBJECTIVE: To investigate the effectiveness of open reduction and internal fixation with locking anatomical plate for treating valgus impacted proximal humerus fracture. METHODS: A retrospective analysis was made on the clinical data of 38 patients with valgus impacted proximal humerus fracture who underwent open reduction and internal fixation with locking anatomical plate between January 2009 and January 2014. There were 25 males and 13 females, aged from 47 to 63 years (mean, 52.3 years); the left and the right sides were involved in 18 and 20 cases, respectively. The causes of fracture included high falling injury in 10 cases, traffic accident injury in 15 cases, and falling injury in 13 cases. The time between injury and operation was 5-10 days (mean, 7.5 days). The collodiaphyseal angle was 160-200 degrees (mean, 176 degrees) on X-ray films. RESULTS: All incisions healed by first intention, and there was no early complication related to operation. All these patients were followed up 12-30 months (mean, 18 months). X-ray film showed that clinical healing time of fracture was 10-16 weeks after operation (mean, 12 weeks); at 12 months after operation, the collodiaphyseal angle recovered to 120-145 degrees (mean, 135 degrees). During follow-up, no loss of fracture reduction and no loosening of internal fixation were observed. At 10-12 months, osteonecrosis of the humeral head occurred in 3 cases (7.9%), including 2 cases of Cruess stage III and 1 case of Cruess stage IV. At last follow-up, the Constant shoulder joint scores were 56-95 (mean, 82.6); the results were excellent in 10 cases, good in 15 cases, fair in 9 cases, and poor in 4 cases, with an excellent and good rate of 66%. Visual analogue scale (VAS) scores were 0-6 (mean, 0.9). CONCLUSION: It can achieve a comparatively satisfactory clinical result to use open reduction and internal fixation with locking anatomical plate for treating valgus impacted proximal humerus fracture.

Recombinant human lactoferrin enhances the efficacy of triple therapy in mice infected with Helicobacter pylori.

Helicobacter pylori (H. pylori) is a life-threatening pathogen which causes chronic gastritis, gastric ulcers and even stomach cancer. Treatment normally involves bacterial eradication; however, this type of treatment only has a rate of effectiveness of <80%. Thus, it is a matter of some urgency to develop new therapeutic strategies. Lactoferrin, a member of the transferrin family of iron-binding proteins, has been proven to be effective in removing a vast range of pathogens, including H. pylori. In the present study, we examined the effectiveness of recombinant human lactoferrin (rhLf) isolated from transgenic goats as a treatment for H. pylori in vitro and in vivo. For the in vivo experiments, BALB/c mice received an intragastric administration of 0.1 ml of a suspension of H. pylori. The mice were then divided into 4 groups: group A, treated with saline; group B, treated with 1.5 g of rhLF; group C, treated with the standard triple therapy regimen; and group D, treated with the standard triple therapy regimen plus.5 g of rhLF. Following sacrifice, the stomach tissues of the mice were histologically examined for the presence of bacteria. For the in vitro experiments, the bacteria were cultured in BHI broth and RT-qPCR and western blot analysis were carried out to determine the mRNA and protein levels of virulence factors (CagA and VacA) in the cultures. Our results revealed that rhLf not only inhibited the growth of H. pylori, but also suppressed the expression of two major virulence factors. Moreover, rhLf markedly increased bacterial eradication and effectively reduced the inflammatory response when combined with the standard triple therapy regimen. These results provide evidence supporting the use of rhLF as an adjuvant to traditional therapeutic strategies in the treatment of H. pylori.

Establishment of a rapid and scalable gene expression system in livestock by site-specific integration.

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.

Large-scale production of functional human lysozyme in transgenic cloned goats.

Human lysozyme (hLZ), an essential protein against many types of microorganisms, has been expressed in transgenic livestock to improve their health status and milk quality. However, the large-scale production of hLZ in transgenic livestock is currently unavailable. Here we describe the generation of transgenic goats, by somatic cell-mediated transgenic cloning, that express large amounts of recombinant human lysozyme (rhLZ) in milk. Specifically, two optimized lysozyme expression cassettes (ß-casein/hLZ and ß-lactoglobulin/hLZ) were designed and introduced into goat somatic cells by cell transfection. Using transgenic cell colonies, which were screened by 0.8 mg/mL G418, as a nuclear donor, we obtained 10 transgenic cloned goats containing one copy of hLZ hybrid gene. An ELISA assay indicated that the transgenic goats secreted up to 6.2 g/L of rhLZ in their milk during the natural lactation period, which is approximately 5-10 times higher than human milk. The average rhLZ expression levels in ß-casein/hLZ and ß-lactoglobulin/hLZ transgenic goats were 2.3 g/L and 3.6 g/L, respectively. Therefore, both rhLZ expression cassettes could induce high levels of expression of the rhLZ in goat mammary glands. In addition, the rhLZ purified from goat milk has similar physicochemical properties as the natural human lysozyme, including the molecular mass, N-terminal sequence, lytic activity, and thermal and pH stability. An antibacterial analysis revealed that rhLZ and hLZ were equally effective in two bacterial inhibition experiments using Staphylococcus aureus and Escherichia coli. Taken together, our experiments not only underlined that the large-scale production of biologically active rhLZ in animal mammary gland is realistic, but also demonstrated that rhLZ purified from goat milk will be potentially useful in biopharmaceuticals.

[Deletion of marker gene in transgenic goat by Cre/LoxP system].

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct.

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3 kb hLF minigene to the regulatory elements of the ß-casein gene. The transgenic goat produced more than 30 mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future.

[Cloning and expression of a single human immunoglobulin heavy-chain variable domain with vascular endothelial growth factor binding activity].

In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.

Fates of donor and recipient mitochondrial DNA during generation of interspecies SCNT-derived human ES-like cells.

To investigate nuclear donor and cytoplast recipient mitochondria fate and their effects on generation of interspecies somatic cell nuclear transfer (iSCNT)-derived human embryonic stem (ES)-like cells, iSCNT embryos were reconstructed between enucleated goat oocytes and human neural stem cells (hNSCs). A total of 10.74% cleaved embryos (13/121) developed to blastocyst stage. One typical primary ES-like (tpES-like) colony and two nontypical primary ES-like (non-tpES-like) colonies designated as non-tpES-like cell-1 and non-tpES-like cell-2, respectively, were obtained from the inner cell masses of iSCNT blastocysts. The tpES-like cells expressed ESC markers. Both human and goat mtDNA could be detected in the embryos at 2-8-, 16-32-cell, and blastocyst stages, and in tpES-like colony and two non-tpES-like colonies. Human mtDNA copies per cell from embryos at two- to eight-cell stage to the three colonies maintain almost its original level, whereas 2.88 x 10(5) goat mtDNA copies per oocyte decreased to 10.8 copies per tpES-like cell, 493 copies per non-tpES-like cell-1, and 77.6 copies per non-tpES-like cell-2, resulting in 43.75% (8.4/19.2), 1.24% (6.2/499), and 14.63% (13.3/90.9) mtDNA content in tpES-like cell, non-tpES-like cell-1, and non-tpES-like cell-2 was that of nuclear donor, respectively. Human-specific Tfam and Polg mRNA could be detected in cells of the three colonies. However, tpES-like colony failed to be passaged. The mRNA level of CoxIV encoded by nuclear donor in tpES-like cell was higher than that in non-tpES-like cell, but significantly lower than that of human ESC, suggesting proper nuclear-cytoplasmic communication would not be established in tpES-like cells. Thus, the data suggest that (1) goat oocytes could reprogram human neural stem cells (hNSCs) into embryonic state and further support the inner cell mass (ICM) of iSCNT blastocyst to form tpES-like colony; (2) nuclear donor mtDNA could be replicated and maintain its original level during the reduction of recipient mitochondrial DNA copies, (3) nuclear-cytoplasmic communication and recipient mtDNA copies might affect the derivation of iSCNT-derived ES-like cells.

Close relatedness between exotic nucleus and cytoplast can improve the postimplantation development rate of cloned intersubspecies embryos.

To improve intersubspecies cloning efficiency, this paper provides six kinds of SCNT embryos with different nuclear-cytoplasmic relatedness to compare the relatedness on the cloning efficiency. Three kinds of SCNT embryos with different relatedness are produced by using Boer goat fibroblast cells as nuclear donors and oocytes of Sannen goat, crossbred F1 (Sannen goat x Boer goat) and Boer goat as cytoplast recipients. Four kinds of SCNT embryos with different relatedness are produced by using Sannen goat oocytes as recipients and fibroblast cells of Boer goat, crossbred F2 (crossbred F1 x Boer goat), crossbred F1, and Sannen goat as nuclear donors. Results show that no obvious differences were observed for preimplantation development of these SCNT embryos. However, different nuclear-cytoplasmic relatedness resulted in obvious differences for postimplantation development of these SCNT embryos. The relatedness is complementary: improving either cytoplasmic compatibility relatedness to nucleus or nuclear relatedness to cytoplast could reduce the gestation loss rate, and increase the birth rate of cloned intersubspecies embryos significantly. But a further amelioration of the relatedness did not improve the postimplantation development in direct proportion. These results suggested that close nuclear-cytoplasmic relatedness can improve the postimplantation development rate of cloned intersubspecies embryos.

Isolation and characterization of human anti-VEGF165 monoclonal antibody with anti-tumor efficacy from transgenic mice expressing human immunoglobulin loci.

The purpose of this study was to prepare a fully human anti-VEGF (vascular endothelial growth factor) monoclonal antibody with anti-tumor activity from five-feature mice which express human immunoglobin loci. Four hybridomas secreting mAb stably were isolated successfully. Some characters such as isotypes, cross-reactivity, inhibition on the binding of hVEGF to VEGFR-2, dissociation constants and the idiotypic characteristic were determined. Proliferation of T24 and Ls-174-T cell line and nude mice bearing human colorectal cancer were used to evaluate therapeutic effects and safety of this mAb. Pharmacokinetics data shows the half life of this mAb was about 5 days after a single intravenous injection. These results suggest the fully human anti-VEGF mAb maybe safe and efficient for cancer treatment.

Production of Prnp-/- goats by gene targeting in adult fibroblasts.

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

[Expression and analysis of the recombinant human interleukin-21 (rhIL-21) in Pichia pastoris].

Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.

Goat MII ooplasts support preimplantation development of embryos cloned from other species.

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.

Excision of selectable genes from transgenic goat cells by a protein transducible TAT-Cre recombinase.

The Cre/loxP site-specific recombination system is a widely used tool for genetic engineering of mammalian genomes. Recombination of loxP-modified alleles is often induced by introduction of foreign DNA vector expressing Cre into the cells. But the introduced DNA vector has the potential to integrate into the genome of the cells and continuous expression of Cre recombinase from the foreign vector has the potential to yield cytotoxicity and genotoxicity in various cells. In this study, we investigate the possibility of overcoming this limitation by using a cell-permeable TAT-Cre recombinase. We found that TAT-Cre treatment of transgenic goat fibroblast cells did not compromise the development competency of reconstructed embryos by using these TAT-Cre-treated cells as nuclear donor in nuclear transfer. Finally, we obtained two live cloned goats where a selectable gene cassette was removed. Our work not only provided an efficient protein transduction-based system for removing selectable genes from transgenic goats, but also presented strong evidence that no severe damage was made to the host cells during the process of protein transduction.

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.

[Targeting Prnp in bovine fibroblasts by promoter-trap strategy].

Promoter-trap strategy for enriching targeted colonies has been usually used to elevate the gene targeting efficiency in somatic cells. Knocking out Prnp in animals by gene targeting can render them resistant to Prion diseases. We constructed a bovine Prnp promoter-less targeting vector BoPrPneo, then transfected the linearized vector into the bovine fetal fibroblasts BFF through electroporation. After selecting in cell culture medium with 250 microg/mL G418, we obtained 99 drug-resistant cell colonies, 4 of them were positive for targeted events after PCR screening, and the targeted colonies were further confirmed by sequencing and Southern blotting. This suggests that one allele of Prnp has been successfully knocked out in bovine fetal fibroblasts. This research supplies a simple, safe and effective method to targeting bovine Prnp.

Expression of active recombinant human lactoferrin in the milk of transgenic goats.

This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in transgenic goats.