Transgenic animals are those in which recombinant DNA technology is used to remove a gene or a specific part of DNA with exogenous DNA, which is hereditable and can be expressed stably. Transgenic rodent technology, first demonstrated by Gordon in 1980,opened the door to the next step in biotechnology and gene regulation, which has been used to reproduce or mimic a part of a human disease in a complex cellular environment by creating in vivo models with germ line transmission of the introduced genetic regulatory elements. Animal models for drug discovery are developing increasingly, which is promoting the research and development new drugs greatly. The use and development of transgenic animals in pharmacological research is reviewed in this article.

Objective To establish a double reporter transgenic mice for monitoring Cre recombinase activity. Methods ZAP DNA fragment with lacZ and human alkaline phosphatase (hAP) gene was microinjected into the male pronucleus of 554 fertilized eggs from C57BL/6 mice. The founder mice and their progeny were screened for integration of transgene into the mouse genome by PCR and Southern blotting. The expression of lacZ transgene at early embryos from F1 generation mice was analyzed by X gal staining. Results A total of 398 survival ZAP DNA injected fertilized eggs were transfered to the oviducts of 21 pseudopregnant recipient mice. Of the 21 recipient mice, 13 became pregnancy and gave birth to 68 offspring mice. The zygote survival rate and birth rate were 71% (398/554) and 17% (68/398), respectively. Of the 68 offspring mice, 9 mice (5 males and 4 females) were identified by PCR and Southern blot analysis. Total integration rate and efficiency of transgene was 13% (9/68) and 1.6% (9/554), respectively. Nine mice as the founders were back crossed to set up F1 generation with other inbred C57BL/6 mice. Out of 9 transgenic mice, transmission of reporter gene in F1 offspring mice followed Mendelian rules, but the expression of lacZ protein was detected at the early embryonic stage (13.5 days postcoitum) in only 3 mice. Conclusion A double reporter transgenic mice for monitoring Cre recombinase activity is established.

The technology of synthesis, extraction and modification of biodegradable polyhydroxybutyrate (PHB) is introduced briefly in this article. It is also summarized that the research progress in application of PHB in drug delivery and tissue engineering.
Biocompatible Materials ; chemistry ; Drug Delivery Systems ; Hydroxybutyrates ; chemistry ; Tissue Engineering

In this article, based on the relative references in the latest 8 years, we introduce briefly the origin, preparation and application of various injectable and absorbable gelling scaffold materials, and outline their advantages and disadvantages as localized gelling scaffold systems.
Absorbable Implants ; Biocompatible Materials ; Gels ; chemistry ; Injections ; Tissue Engineering ; methods

Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method.Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution.The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test.Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker.Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body.Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity.After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype.Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.

Objective To investigate the expression of TROP2 and MMP-9 protein expression in cholangiocarcinomas and their relationship between the pathological behavior and prognosis.Methods A total of 54 patients who were diagnosed with cholangiocarcinoma in the People's Hospital of Binzhou,were retrospectively reviewed.Immunohistochemical staining and Log rank test were used to detect the expression of TROP2 and MMP-9 protein in 54 cases of cholangiocarcinomas and 18 cases of normal bile duct tissues achieved by partial hepatectomy of hepatolithiasis.Results The positive expression rate of TROP2 in cholangiocarcinoma tissues (55.6％) was higher than that of normal bile duct tissues (5.6％).The positive expression rate of MMP-9 in cholangiocarcinoma tissues (51.9％) was higher than that of normal bile duct tissues (11.1％).The differences of the expression of TROP2 and MMP-9 in cholangiocarcinoma of TNM stage,lymph node metastasis and neural invasion were significant(all P ＜ 0.05).There was significant positive correlation between TROP-2 and MMP-9 expression by using spearman correlation analysis (r =0.555,P ＜ 0.001).Survival analysis showed that TROP2 expression was an independent prognostic factor in cholangiocarcinoma.Conclusions TROP2 plays a important role in the development and metastasis of cholangiocarcinoma.Thus,TROP2 may be a prognostic indicator for cholangiocarcinoma.

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.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Organism ; veterinary ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors ; genetics ; Goats ; genetics ; Integrases ; chemistry ; metabolism ; Recombination, Genetic ; Transgenes ; genetics

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.
Electroporation ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; analysis ; biosynthesis ; genetics

Objective To evaluate the therapeutic efficacy and safety of transcatheter closure of atrial septal defect (ASD) and patent ductus arteriosus (PDA) using Amplatzer occluder. Methods Routine cardiac catheterization and angiography were performed in 50 patients (23 male, 27 female, age ranging from 3 to 64 years old), including 19 cases of ASD and 31 cases of PDA under local or general anesthesia. After balloon sizing of the ASD, the optimal Amplazter septal occluder (ASO) was transmitted into the left atrial, and the left and right disks were released in turn. The Amplatzer occluder was completely released after transthoracic echocardiography confirmed that there was no residual shunts or new onset mitral valve regurgitation. The Amplatzer duct occluder (ADO) size was selected according to the narrowest point of PDA measured by angiography, and the occluder was released after the repeated angiography showed no residual shunts. Results ① The mean diameter of the ASD measured by balloon was 13-31 (23?6) mm and the diameter of ASO was (17-40) mm. The immediate closure rate was 100%. ② Angiography confirmed that closure of the ductus using ADO was achieved in 30 patients, and closure of the large size (12 mm) was achieved in 1 case of PDA patient using ASO (17 mm). No complications were encountered. Conclusion Transcatheter closure of ASD and PDA using Amplatzer device, with the advantages of simple operation, confirmative occlusion efficacy, minimal invasiveness, wide indications, and less complications, has a bright future of clinical application.

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.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; metabolism