Generating Goat Mammary Gland Bioreactors for Producing Recombinant Proteins by Gene Targeting.

Exogenous genes can be site-specifically integrated into the genomic DNA of animals by homologous recombination, generating transgenic animals. These animals have a clear hereditary background, although position effects of the exogenous genes and potential functional disruption of host genes can be caused by the genetic inserts. Therefore, the generation of mammary gland bioreactors via gene-targeting methods is a great asset for producing recombinant proteins in milk. Here, we describe a method of generating gene-targeted goats with the human alpha-lactalbumin gene (hα-LA) integrated into the beta-lactoglobulin gene (BLG) locus. The milk from these goats will be less allergenic and will be enriched with components of human milk protein.

Balloon-expandable stent angioplasty in the treatment of vertebral artery stenosis in the V2 segment.

INTRODUCTION: Vertebral artery stenosis is a major cause of posterior circulation ischemia in the elderly. There is not a clear consensus on the optimal therapeutic approach for symptomatic extracranial vertebral artery stenosis. AIM: To evaluate the feasibility and efficacy of balloon-expandable stent angioplasty in the treatment of vertebral artery stenosis in the V2 segment. MATERIAL AND METHODS: Five patients with vertebral artery stenosis (V2 segment) and treatment of percutaneous transluminal stenting from July 2009 to June 2014 were retrospectively evaluated. All patients underwent color Doppler, transcranial color Doppler (TCD), CT angiography (CTA) and cerebral digital subtraction angiography (DSA) preoperatively. Whether there was osseous oppression was determined according to neck computed tomography (CT) and CTA. After the surgery, angiography was performed to determine if there was infarction or bleeding in the intracranial vertebral artery, basilar artery and posterior cerebral artery. The surgical parameters, residual stenosis, complications, etc. were recorded and evaluated. The patients were followed up accordingly. RESULTS: Five patients (3 males, 2 females; average age of 66 ±4.2, range of 54-75) were enrolled in the study. Balloon-expandable stents were successfully implanted in the 5 patients. The mean residual stenosis after the balloon-expandable stenting (preoperative: average, 87.0 ±6.6%, range: 75-93%) was 12.6 ±7.8% (range: 5-25%). The clinical symptoms disappeared or receded. No serious complications occurred. CONCLUSIONS: The balloon-expandable stent angioplasty seemed to be feasible and efficacious in treating vertebral artery stenosis in the V2 segment. Further study with a large sample size is needed.

Computational method for correcting complex optical distortion based on FOV division.

We propose a computational method for correcting complex optical distortion in off-axis optical systems, such as the optical systems found in head-mounted and head-up displays. The proposed method divides the wide field of view (FOV) into subsections, thereby allowing the distortion to be calculated for each small FOV. Instead of applying the conventional distortion model, the distortion coefficients for each small FOV can be calculated using a simple linear polynomial. In addition, in contrast to the conventional distortion coefficients that refer to the deviation between the real and paraxial image, the distortion coefficients employed by this method directly characterize the relationship between the object and its image. Thus, using the polynomial in the reverse manner repeatedly for each small FOV with the corresponding distortion coefficients, a pixel lookup table is obtained, which can be used to accurately compensate for the distortion in the optical system. This method avoids complicated computations, and there are no requirements for intrinsic or extrinsic parameters. Our experiments verified the effectiveness of the method where the root-mean-square deviation of the projected distorted straight lines was corrected from 23 to 65 pixels to approximately 1 pixel.

Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin.

BACKGROUND: Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein. RESULTS: Secretion level of the fusion protein produced in vitro in BHK cells was approximately 30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04-1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of approximately 150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (approximately 32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (approximately 3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested. CONCLUSION: Both the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.

Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning.

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat beta-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.

Production and characterization of recombinant equine prorelaxin.

Relaxin is a peptide hormone produced by a wide variety of mammals. In the horse, the placenta is the major source of relaxin. Since pure equine relaxin is difficult to obtain to study its role in the pregnant mare, the objectives of this study were to produce recombinant equine prorelaxin and characterize its immunological and biological activity. First, an equine relaxin gene cassette was transfected into immortalized bovine mammary epithelial (MAC-T) cells. Second, immunological activity of media conditioned by transfected MAC-T cells was tested by Western blotting and quantified using a homologous equine radioimmunoassay. Finally, bioactivity of the conditioned media was tested using the human monocyte cell line, THP-1, which exhibits a rapid and dose-dependent increase in the accumulation of cAMP upon binding relaxin. The results showed that conditioned media, concentrated 5x, yielded 4.11 +/- 0.81 ng/ml recombinant equine prorelaxin. In addition, a 19 kDa immunoreactive band, corresponding to the expected size of equine prorelaxin, was visualized by SDS-PAGE. THP-1 cells incubated with conditioned media (5x) from transfected cells, in the presence of forskolin (1 microM) and isobutylmethylxanthine (50 microM), showed an increase in cAMP production over media from mock-transfected cells alone. In conclusion, recombinant equine prorelaxin secreted by MAC-T cells was both immunologically and biologically active. This study demonstrates the first attempt to produce recombinant equine prorelaxin, important for further study of the role of relaxin in the mare.

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice.

BACKGROUND: Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. RESULTS: The GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney. CONCLUSION: The goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals.