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Predator Stress Can Exert Detrimental Effects on Female Mammals, Leading to Disrupted Reproduction. Although Many Studies Have Addressed the Effects of Predator Stress on Reproductive Output in Rodents, Few Studies Have Focused on the Effect of Visual or Auditory Stress on Pregnant Females. in This Study, We Investigated the Possible Effect of Predator Stress, Either Visual Only or Combined Visual and Auditory (visual+auditory), on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer. Reproductive Performance Was Assessed As Pregnancy Rate, Implantation Rate, Gestation Length, Live Pup Rate, and Neonatal Birth Weight. Moreover, Serum Cortisol and Progesterone Levels in Dams Were Measured by Using Electrochemiluminescence Immunoassay. Exposure to Predator (cat) Stress Did Not Lead to a Significant Change in Pregnancy Rates in the Tested Mice. However, the Stressed Mice Showed Significantly Decreased Implantation Rates Compared with the Control Group. Similarly, the Live Pup Rate and Neonatal Birth Weight Were Significantly Lower in the Group Exposed to Preda- Tor Stress Than in the Control Group. Furthermore, Mice Exposed to Visual+auditory Stress Showed a Significant Reduction in Gestation Length Compared with the Control Mice. Our Data Showed That Predator Visual+auditory Stress As Combined Stimuli Significantly Increased Serum Cortisol Level. in Contrast, Progesterone Levels Did Not Significantly Vary Among the Experimental Groups. Taken Together, Our Findings Imply That Predator Stress Adversely Affects the Reproductive Efficiency of Pregnant Mice By Decreasing the Implantation Rate, Live Birth Rate, and Neonatal Birth Weight and by Prolonging Gestation Length.

Background Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS. Methods CRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed. Results Six CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals. Conclusion A CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.

Additional file 1: Table S1. The sequences of potential off-target sites.

Additional file 2: Table S2. The sequences of potential off-target loci PCR primers.

There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce transgenic cloned goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing goat β-casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25-rhLF-Neo vector was microinjected into goat fetal fibroblasts. G418 selection and PCR analysis were used to identify transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this transgenic goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high-purity (95.8%) rhLF from a cation-exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a transgenic goat harboring the hLF transgene driven by the goat β-casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.

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.

Objective To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

Methyltrioxorhenium (MTO) has been applied as catalyst to promote cellulose hydrolysis by using the ionic liquid 1-allyl-3-methylimidazolium chloride ([Amim]Cl) as solvent. When using 7 mol% of MTO, 70 μL of water, ca. 0.6 mmol of microcrystalline cellulose and 2.0 g of [Amim]Cl under microwave irradiation for 30 min at 150 °C, 51.2% of total reducing sugar (TRS) and 24.7% of glucose yield can be obtained. The nucleophilic attack of electron-rich O atom of β-1,4-glycosidic bond to electron-poor Re atom of MTO, leading to the broken of β-1,4-glycosidic bond, is assumed to be crucial for cellulose degradation.

Targeted mutagenesis by zinc-finger nucleases (ZFNs) can be used to generate knock-out mammalian cell lines with high efficiency. A number of different methods have been developed for the design and assembly of gene-specific ZFNs, making them easily accessible to researchers. In this study, we used ZFNs assembled through the CoDA (context-dependent assembly) platforms to generate mutant caprine fetal fibroblasts cells for the BLG gene. ZFN plasmid was introduced into the caprine fetal fibroblasts cell by electroporation. ZFN-induced cleavage of the target sequence was confirmed by the Surveyor nuclease assay analysis. Sequence analysis revealed that ZFN-induced mutations such as base insertion, deletion, or substitution were generated in the ZFN cleavage site of BLG. The simplicity and efficacy of CoDA will enable broad application of ZFN technology. This technique could be used with homologous arm, which may target foreign genes into the BLG locus at higher efficiency.

Crowding is a key factor in tourists’ experience in theme parks, and mitigating crowding makes parks more competitive. This study examines how to effectively mitigate crowding in theme parks. First, a Markov-based method is developed to predict the spatial-temporal distribution of tourists in the park. Then, a prospective coordination approach based on the tourist distribution prediction is proposed. To evaluate the performance of this approach, an experiment is constructed using an agent-based simulation platform. The results indicate that the proposed method significantly outperforms existing methods. Furthermore, we conduct two experiments and, based on the results, offer several recommendations for crowd management.

This study evaluates the relationship between the genotype and milk protein components in goats. Milk samples were collected from cloned goats and normal white goats during different postpartum (or abortion) phases. Two cloned goats, originated from the same somatic line of goat mammary gland epithelial cells, and three sexually reproduced normal white goats with no genetic relationships were used as the control. The goats were phylogenetically analyzed by polymerase chain reaction-restriction fragment length polymorphism. The milk protein components were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicated that despite the genetic fingerprints being identical, the milk protein composition differed between the two cloned goats. The casein content of cloned goat C-50 was significantly higher than that of cloned goat C-4. Conversely, although the genetic fingerprints of the normal white goats N-1, N-2, and N-3 were not identical, the milk protein profiles did not differ significantly in their milk samples (obtained on postpartum day 15, 20, 25, 30, and 150). These results indicated an association between milk protein phenotypes and genetic polymorphisms, epigenetic regulation, and/or non-chromosomal factors. This study extends the knowledge of goat milk protein polymorphisms, and provides new strategies for the breeding of high milk-yielding goats.

Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary‑specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme‑linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was ~950 µg/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.

Due to unbalanced distribution of tourists during peak time, tourists are likely to highly flock in certain areas of scenery spots if imperfect organization and management. For this reason, there may be crowds or even trample accidents in addition to unrecoverable damages for the ecological environment in such areas. Therefore, the valid solution is to formulate scientific tourist diversion strategy, reasonably adjust the tour routes, and realize the balanced distribution of tourists over space and time. The prerequisite and base of the research is to establish scientific rational evaluation indicators for dive rsion strategy. At present, the common indicators include the Variance Model reflecting the balance of load rate for each spot in the sc enic region and the Gini-Simpson Index (G-S Index) showing the dispersion degrees of tourists among the scenery spots. However, these indexes highlight the opinions of scenery region managers but ignore the tourism utility during the travel. Such indexes can be used to evaluate the diversion strategy only from a macro view but hardly facilitates shaping any scientific valid suggestions for plan impro vement. So this thesis established the evaluation model of tourism utility function (TUF) based on the spot congestion degree and compared the simulation effects of three diversion strategies respectively with three evaluation models, including Variance, G-S Index and TUF. It is found from the comparison results that TUF can better the overall evaluation of the diversion strategies and reveal th e relevant inherent problems so as to provide reasonable and suggestions for scientific management on the scenery spot.

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column. Results: There were 12 hybridoma strains selected into the polyol responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA. Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins. Keywords: tPA, Immunoaffinity chromatography, PR-mAb, ELISA-elution, Antibody, Thrombolytic activity

TALEN target sequences. (DOC 35Â kb)

Off-target sites assayed for MTAL-2. (DOC 83Â kb)

Primer pairs for PCR amplification of off-target regions for MTAL-2. (DOC 32Â kb)

Primers for PCR amplification of TALENs target regions. (DOC 29Â kb)

Background Myostatin (MSTN) encodes a negative regulator of skeletal muscle mass that might have applications for promoting muscle growth in livestock. In this study, we aimed to test whether targeted MSTN editing, mediated by transcription activator-like effector nucleases (TALENs), is a viable approach to create myostatin-modified goats (Capra hircus). Results We obtained a pair of TALENs (MTAL-2) that could recognize and cut the targeted MSTN site in the goat genome. Fibroblasts from pedigreed goats were co-transfected with MTAL-2, and 272 monoclonal cell strains were confirmed to have mono- or bi-allelic mutations in MSTN. Ten cell strains with different genotypes were used as donor cells for somatic cell nuclear transfer, which produced three cloned kids (K179/MSTN−/−, K52-2/MSTN+/−, and K52-1/MSTN+/+). Conclusions The results suggested that the MTAL-2 could disrupt MSTN efficiently in the goat genome. The mutated somatic cells could be used to produce MSTN-site mutated goats without developmental disruption. Thus, TALENs is an effective method for accurate genome editing to produce site-modified goats.

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 15.2–630 µg/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.

Quaternary ammonium perrhenates were applied as catalyst to promote the hydrolysis of cellulose in 1-allyl-3-methylimidazolium chloride ([Amim]Cl). The quaternary ammonium perrhenates displayed good catalytic performance for cellulose hydrolysis. Water was also proven to be effective to promote cellulose hydrolysis. Accordingly, 97% of total reduced sugar (TRS) and 42% of glucose yields could be obtained under the condition of using 5mol% of tetramethyl ammonium perrhenate as catalyst, 70μL of water, ca. 0.6mmol of microcrystalline cellulose (MCC) and 2.0g of [Amim]Cl as solvent under microwave irradiation for 30min at 150°C (optimal conditions). The influence of quaternary ammonium cation on the efficiency of cellulose hydrolysis was examined based on different cation structures of perrhenates. The mechanism on perrhenate catalyzed cellulose hydrolysis is also discussed, whereas hydrogen bonding between ReO4 anion and hydroxyl groups of cellulose is assumed to be the key step for depolymerization of cellulose.

Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.

The doctor’s performance can be divided into two kinds of task performance and contextual performance, and current articles on contextual performance are less. This paper is studied by Team Principal-agent theory. The benchmark mode is established for contrast, in which agents don’t make efforts on contextual performance. And then, according to whether accounting cost respectively, we set up two models that only one agent has contextual performance. The conclusion shows contextual performance is obvious in medical institutions, and it benefits to the whole team. Therefore, we advocate using team performance compensation plan, and then explain the necessity of government subsidies for medical institutions.

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

In this study, we evaluated the development potential of caprine mammary gland epithelial cells (CMGECs) after transfection and nuclear transfer into enucleated, ovulated oocytes. We first isolated CMGECs from udders of lactating goats which were transfected with expression plasmid for human lacterrin and selected by G418. Then we chose sixteen neomycin resistant lines and induced them with prolactin for the expression of human lactoferrin checked by Western blotting. The donor cells, expressing human lactoferrin of 75 kD, were fused and activated with enucleated ovulated oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 16 recipient goats. There were fourteen (87.5%), thirteen (81.3%), and ten (62.5%) pregnancies confirmed pregnant by ultrasound on Day 30, 60, and 90, respectively. Three recipients carried the pregnancies to term and delivered one goat each. Nested PCR-RFLP analysis confirmed that all of the kids were clones of the donor cells. These results demonstrated that CMGECs after transfection remain totipotent for nuclear transfer.