Impact of telomere length and mitochondrial DNA copy number variants on survival of newborn cloned calves.

An established technology to create cloned animals is through the use of somatic cell nuclear transfer (SCNT), in which reprogramming the somatic cell nucleus to a totipotent state by enucleated oocyte cytoplasm is a necessary process, including telomere length reprogramming. The limitation of this technology; however, is that the live birth rate of offspring produced through SCNT is significantly lower than that of IVF. Whether and how telomere length play a role in the development of cloned animals is not well understood. Only a few studies have evaluated this association in cloned mice, and fewer still in cloned cows. In this study, we investigated the difference in telomere length as well as the abundance of some selected molecules between newborn deceased cloned calves and normal cows of different ages either produced by SCNT or via natural conception, in order to evaluate the association between telomere length and abnormal development of cloned cows. The absolute telomere length and relative mitochondrial DNA (mtDNA) copy number were determined by real-time quantitative PCR (qPCR), telomere related gene abundance by reverse-transcription quantitative PCR (RT-qPCR), and senescence-associated ß-galactosidase (SA-ß-gal) expression by SA-ß-gal staining. The results demonstrate that the newborn deceased SCNT calves had significantly shortened telomere lengths compared to newborn naturally conceived calves and newborn normal SCNT calves. Significantly lower mtDNA copy number, and significantly lower relative abundance of LMNB1 and TERT, higher relative abundance of CDKN1A, and aberrant SA-ß-gal expression were observed in the newborn deceased SCNT calves, consistent with the change in telomere length. These results demonstrate that abnormal telomere shortening, lower mtDNA copy number and abnormal abundance of related genes were specific to newborn deceased SCNT calves, suggesting that abnormally short telomere length may be associated with abnormal development in the cloned calves.

Therapeutic Effects of Hematopoietic Stem Cell Derived From Gene-Edited Mice on ß654-Thalassemia.

ß-thalassemia is an inherited blood disease caused by reduced or inadequate ß-globin synthesis due to ß-globin gene mutation. Our previous study developed a gene-edited mice model (ß654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the ßIVS2-654 (C > T) mutation site and the 3' splicing acceptor site at 579 and corrected abnormal ß-globin mRNA splicing in the ß654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from ß654-ER mice on ß-thalassemia by consecutive HSC transplantation. The results indicated that HSC transplantation derived from gene-edited mice can significantly improve the survival rate of mice after lethal radiation doses and effectively achieve hematopoietic reconstruction and long-term hematopoiesis. Clinical symptoms, including hematologic parameters and tissue pathology of transplanted recipients, were significantly improved compared to the non-transplanted ß654 mice. The therapeutic effect of gene-edited HSC transplantation demonstrated no significant difference in hematological parameters and tissue pathology compared with wild-type mouse-derived HSCs. Our data revealed that HSC transplantation from gene-edited mice completely recovered the ß-thalassemia phenotype. Our study systematically investigated the therapeutic effect of HSCs derived from ß654-ER mice on ß-thalassemia and further confirmed the efficacy of our gene-editing approach. Altogether, it provided a reference and primary experimental data for the clinical usage of such gene-edited HSCs in the future.

Loss of Protein Function Causing Severe Phenotypes of Female-Restricted Wieacker Wolff Syndrome due to a Novel Nonsense Mutation in the ZC4H2 Gene.

Pathogenic variants of zinc finger C4H2-type containing (ZC4H2) on the X chromosome cause a group of genetic diseases termed ZC4H2-associated rare disorders (ZARD), including Wieacker-Wolff Syndrome (WRWF) and Female-restricted Wieacker-Wolff Syndrome (WRWFFR). In the current study, a de novo c.352C>T (p.Gln118*) mutation in ZC4H2 (NM_018684.4) was identified in a female neonate born with severe arthrogryposis multiplex congenita (AMC) and Pierre-Robin sequence (cleft palate and micrognathia). Plasmids containing the wild-type (WT), mutant-type (MT) ZC4H2, or GFP report gene (N) were transfected in 293T cell lines, respectively. RT-qPCR and western blot analysis showed that ZC4H2 protein could not be detected in the 293T cells transfected with MT ZC4H2. The RNA seq results revealed that the expression profile of the MT group was similar to that of the N group but differed significantly from the WT group, indicating that the c.352C>T mutation resulted in the loss of function of ZC4H2. Differentially expressed genes (DEGs) enrichment analysis showed that c.352C>T mutation inhibited the expression levels of a series of genes involved in the oxidative phosphorylation pathway. Subsequently, expression levels of ZC4H2 were knocked down in neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) by lentiviral-expressed small hairpin RNAs (shRNAs) against ZC4H2. The results also demonstrated that decreasing the expression of ZC4H2 significantly reduced the growth of NSCs by affecting the expression of genes related to the oxidative phosphorylation signaling pathway. Taken together, our results strongly suggest that ZC4H2 c.352C>T (p.Gln118*) mutation resulted in the loss of protein function and caused WRWFFR.

Spatial transcriptomic profiling to identify mesoderm progenitors with precision genomic screening and functional confirmation.

OBJECTIVES: Mesoderm, derived from a new layer between epiblast and hypoblast during gastrulation, can differentiate into various tissues, including muscles, bones, kidneys, blood, and the urogenital system. However, systematic elucidation of mesoderm characteristics and specific markers remains a challenge. This study aims to screen and identify candidate genes important for mesoderm development. MATERIALS AND METHODS: Cells originating from the three germ layers were obtained by laser capture microdissection, followed by microcellular RNA sequencing. Mesoderm-specific differentially expressed genes (DEGs) were identified by using a combination of three bioinformatics pipelines. Candidate mesoderm-specific genes expression were verified by real-time quantitative polymerase chain reaction analysis and immunohistochemistry. Functional analyses were verified by ESCs-EBs differentiation and colony-forming units (CFUs) assay. RESULTS: A total of 1962 differentially expressed mesoderm genes were found, out of which 50 were candidate mesoderm-specific DEGs which mainly participate in somite development, formation of the primary germ layer, segmentation, mesoderm development, and pattern specification process by GO analysis. Representative genes Cdh2, Cdh11, Jag1, T, Fn-1, and Pcdh7 were specifically expressed in mesoderm among the three germ layers. Pcdh7 as membrane-associated gene has hematopoietic-relevant functions identified by ESCs-EBs differentiation and CFUs assay. CONCLUSIONS: Spatial transcriptomic profiling with multi-method analysis and confirmation revealed candidate mesoderm progenitors. This approach appears to be efficient and reliable and can be extended to screen and validate candidate genes in various cellular systems.

Maternal Prkce expression in mature oocytes is critical for the first cleavage facilitating maternal-to-zygotic transition in mouse early embryos.

OBJECTIVES: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal-to-zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high- or low- competence mouse strains. MATERIALS AND METHODS: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real-time quantitative PCR (RT-qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment. RESULTS: The fertilization and 2-cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty-nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT-qPCR. Prkce knockout mice showed a reduced 2-cell rate (Prkce+/+ 80.1% vs. Prkce-/- 32.4%) that could be rescued by Prkce cRNA injection (2-cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation. CONCLUSIONS: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2-cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high-quality mature oocytes.

Correction of RNA splicing defect in ß654-thalassemia mice using CRISPR/Cas9 gene-editing technology.

ß654-thalassemia is a prominent Chinese subtype of b-thalassemia, representing 17% of all cases of ß-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant ß-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73- nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant b-globin RNA splicing and ameliorate the clinical ß-thalassemia syndrome in ß654 mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of ß654 or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited ß654 founders and their offspring were significantly improved compared to those of the non-edited ß654 mice, consistent with the restoration of wild-type b-globin RNA expression. Notably, the survival rate of gene-edited heterozygous ß654 mice increased significantly, and liveborn homozygous ß654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of ß654-thalassemia therapy in the future.

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient.

Down's syndrome (DS) is one of the most commonly known disorders with multiple congenital disabilities. Besides severe cognitive impairment and intellectual disability, individuals with DS also exhibit additional phenotypes of variable penetrance and severity, with one or more comorbid conditions, including Alzheimer's disease, congenital heart disease, or leukemia. Various vital genes and regulatory networks had been studied to reveal the pathogenesis of the disease. Nevertheless, very few studies have examined alternative splicing. Alternative splicing (AS) is a regulatory mechanism of gene expression when making one multi-exon protein-coding gene produce more than one unique mature mRNA. We employed the GeneChip Human Transcriptome Array 2.0 (HTA 2.0) for the global gene analysis with hiPSCs from DS and healthy individuals. Examining differentially expressed genes (DEGs) in these groups and focusing on specific transcripts with AS, 466 up-regulated and 722 down-regulated genes with AS events were identified. These genes were significantly enriched in biological processes, such as cell adhesion, cardiac muscle contraction, and immune response, through gene ontology (GO) analysis of DEGs. Candidate genes, such as FN1 were further explored for potentially playing a key role in DS. This study provides important insights into the potential role that AS plays in DS.

Monitoring the authenticity of pu'er tea via chemometric analysis of multielements and stable isotopes.

Mineral elements and stable isotopes combined with stoichiometric methods were used as a potential tool for first authenticating Chinese tea according to it's production year. A total of 86 mineral elements and stable isotope compositions were determined from the Xiangzhujing Pu'er tea in five different production years using ICP-MS and ICP-OES. On the basis of 78 statistically significant mineral elements and stable isotopes, HCA, PCA, PLS-DA, BP-ANN, and LDA were employed to build authentication models for predicting the Pu'er tea with different production years. The clustering results of the HCA and PCA were worse than that of PLS-DA, BP-ANN, and LDA. The PLS-DA model displayed a perfect model performance (R2X = 0.86, R2Y = 0.974, and Q2 = 0.922). The authentication performance of LDA and BP-ANN revealed their 100% recognition sensitivity and prediction ability and was thus better than that of PLS-DA. Mn, 68Zn, and 203Tl were the markers for enabling the successful authentication of Pu'er tea with different production years. This study contributes toward generalizing the use of mineral element and stable isotope fingerprinting combined with LDA and BP-ANN as a promising tool for authentication of tea worldwide.

Improved geographical origin discrimination for tea using ICP-MS and ICP-OES techniques in combination with chemometric approach.

BACKGROUND: There is an urgent need to strengthen the testing and certification of geographically iconic foods, as well as to use discriminatory science and technology for their regulation and verification. Multi-element and stable isotope analyses were combined to provide a new chemometric approach for improving the discrimination tea samples from different geographical origins. Different stoichiometric methods [principal component analysis (PCA), hierarchical cluster analysis (HCA), partial least squares-discriminant analysis (PLS-DA), back propagation based artificial neural network (BP-ANN) and linear discriminant analysis (LDA)] were used to demonstrate this discrimination approach using Yongchuanxiuya tea samples in an experimental test. RESULTS: Multi-element and stable isotope analyses of tea samples using inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry easily distinguished the geographical origins. However, the clustering ability of the two unsupervised learning methods (PCA and HCA) were worse compared to that of the three supervised learning methods (PLS-DA, BP-ANN and LDA). BP-ANN and LDA, with 100% recognition and prediction abilities, were found to be better than PLS-DA. 86 Sr and 112 Cd were the markers enabling the successful classification of tea samples according to their geographical origins. Under the validation by 'blind' dataset, the prediction accuracies of the BP-ANN and LDA methods were all greater than 90%. The LDA method showed the best performance, with an accuracy of 100%. CONCLUSION: In summary, determination of mineral elements and stable isotopes using inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry techniques coupled with chemometric methods, especially the LDA method, is a good approach for improving the authentication of a diverse range of tea. The present study contributes toward generalizing the use of fingerprinting mineral elements and stable isotopes as a promising tool for testing the geographic roots of tea and food worldwide. © 2020 Society of Chemical Industry.

Production of biologically active human factor IX-Fc fusion protein in the milk of transgenic mice.

OBJECTIVE: To investigate the feasibility of producing human IgG1 Fc fragment fused factor IX (FIX-Fc) in the milk of transgenic animals, for an alternative possible solution to the unmet need of FIX-Fc products for hemophilia B treatment. RESULTS: Six founder lines of transgenic mice harboring FIX-Fc cassette designed to be expressed specifically in the mammary gland were generated. FIX-Fc protein was secreted into the milk of transgenic mice with preserved biological activity (with the highest value of 6.2 IU/mL), similar to that of the non-fused FIX transgenic milk. RT-PCR and immunofluorescence analysis confirmed that FIX-Fc was specifically expressed in the mammary gland. The blood FIX clotting activities were unchanged, and no apparent health defects were observed in the transgenic mice. Moreover, the stability of FIX protein in milk was increased by the Fc fusion. CONCLUSIONS: It is feasible to produce biologically functional FIX-Fc in the mammary gland of transgenic mice. Our preliminary results provide a foundation for the potential scale-up production of FIX-Fc in the milk of dairy animals.

Chemometric authentication of Pu'er teas in terms of multielement stable isotope ratios analysis by EA-IRMS and ICP-MS.

In this work, the stable isotope ratios of carbon, nitrogen, hydrogen, oxygen, and mineral elements and their stoichiometric methods were examined as possible factors that could certify Chinese tea based on its production years. A total of 43 multi-element stable isotope ratios of Xiangzhujing Pu'er tea in five production years were determined through inductively coupled plasma mass spectrometry (ICP-MS) and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) methods. Two unsupervised learning techniques (principal component analysis and hierarchical clustering analysis) and three supervised learning techniques (partial least squares discriminant analysis [PLS-DA], back-propagation artificial neural network [BP-ANN], and linear discriminant analysis [LDA]) were used on the basis of 18 statistically significant multi-elemental stable isotope ratios to build authentication models for Pu'er tea. The clustering abilities of the two unsupervised learning methods were worse than those of the three supervised learning methods. The three supervised models correctly separated the corresponding production years of the samples. The authentication performance was obtained through BP-ANN and LDA, with 100% recognition and prediction abilities, which were better than those of PLS-DA. δD, δ13C, and 154Sm/152Sm were determined as the markers for the accurate authentication of Pu'er tea in different production years. The profiles of multi-element stable isotope ratios obtained via ICP-MS and EA-IRMS with chemometric methods could serve as potential and powerful factors for authenticating Chinese tea in different production years. This study contributed to the generalization of the use of multi-elemental stable isotope ratio fingerprinting as a promising tool for testing the authenticity of tea worldwide.

Improved biosafety of a lentiviral vector by reducing cellular gene activation.

BACKGROUND: Lentiviral vectors (LVs) have enhancer activity and/or transcriptional read-through (EATRT) properties that can lead to the activation of adjacent genes. Consequently, patients may be at increased risk for adverse effects if such vectors are used clinically. METHODS: In the present study, we assessed the abilities of different "pro-LV"-like constructs with respect to decreasing its EATRT, including the "pro-LV" vector bearing a chimeric ΔLTR of the human foamy virus R-U5 region replaced by that of an LV (HF). RESULTS: By analyzing the EATRT of "pro-LV" constructs transfected in 293T cells, we observed that the inclusion of the first 400 bp of the chicken ß-globin locus HS4 insulator core sequence oriented in the reverse direction (C-) combined with two copies of the simian virus 40 upstream-sequence element (U) at the ΔU3 of ΔLTR region of "pro-LV" tended to shield the adjacent genomic sequences, such that the EATRT rate was lower than when either of the C- or U was included in the "pro-LV". Moreover, upon transduction, the pro-HF appears to reduce the EATRT rate in the chromosomes of 293T (by 80%) and human peripheral blood mononuclear cells (PBMCs) (by 75%) compared to when pro-LV C-U was included (with a 60% and 89% reduction in 293T and PBMCs, respectively). The HF construct had a significant reduction of viral biological titer compared tiowhen the pro-LV C-U was used in 293T cells. CONCLUSIONS: The results of the present study provide an important basis for the clinical applicability of LVs in gene and cell therapy.

Efficient stabilization of recombinant human coagulation factor VIII in the milk of transgenic mice using hFVIII and vWF co-expression vector transduction.

OBJECTIVES: To investigate the reasons for the instability of human coagulation factor FVIII (hFVIII) in milk which is an intractable obstacle during the hFVIII production by a transgenic mammary gland bioreactor. RESULTS: We constructed P1A3-hFVIIIBDD and P1A3-hFVIIIBDD-IRES-vWF co-expression cassettes for generating transgenic mice. P1A3-hFVIII/CMV-vWF double heterozygotes were also prepared by mating P1A3-hFVIIIBDD with CMV-vWF mice. hFVIII bioactivity in milk was determined under different storage conditions. The half-life (in vitro) of hFVIII bioactivity in P1A3-hFVIIIBDD-IRES-vWF mice was significantly longer than P1A3-hFVIIIBDD mice [77 ± 4.9 vs. 44 ± 2.6 h at 4 °C, 32.5 ± 5 vs. 19.7 ± 0.6 h at room temperature and 7.4 ± 1.4 vs. 3.4 ± 0.6 at 37 °C, respectively (P < 0.05)]. The half-life (in vitro) of hFVIII bioactivity in milk of double heterozygotes was similar to P1A3-hFVIIIBDD-IRES-vWF ones, demonstrating that the vWF transgene expression in hFVIII transgenic mice can efficiently improve the stabilization of hFVIII bioactivity in milk. CONCLUSION: We provide a new approach of P1A3-hFVIIIBDD-IRES-vWF co-expression to generate more stable hFVIII in transgenic milk with rapid and low cost as well as valuable information for producing pharmaceutical proteins by transgenic mammary gland bioreactor.

Safety assessment of the biogenic amines in fermented soya beans and fermented bean curd.

To evaluate the safety of biogenic amines, high performance liquid chromatography (HPLC) was used to evaluate the levels of biogenic amines in fermented soya beans and fermented bean curd. In fermented soya beans, the total biogenic amines content was in a relatively safe range in many samples, although the concentration of histamine, tyramine, and ß-phenethylamine was high enough in some samples to cause a possible safety threat, and 8 of the 30 samples were deemed unsafe. In fermented bean curd, the total biogenic amines content was more than 900 mg/kg in 19 white sufu amples, a level that has been determined to pose a safety hazard; putrescine was the only one detected in all samples and also had the highest concentration, which made samples a safety hazard; the content of tryptamine, ß-phenethylamine, tyramine, and histamine had reached the level of threat to human health in some white and green sufu samples, and that may imply another potential safety risk; and 25 of the 33 samples were unsafe. In conclusion, the content of biogenic amines in all fermented soya bean products should be studied and appropriate limits determined to ensure the safety of eating these foods.

Applying a resting operation to alleviate bioclogging in vertical flow constructed wetlands: an experimental lab evaluation.

The aim of this study is to evaluate the effects of and analyze the reasons for applying a resting operation to alleviate bioclogging in vertical flow constructed wetlands (VFCWs). In parallel, three groups of laboratory-scale VFCWs were continuously fed with prepared wastewater (BOD = 600 mg/L) at a relatively high hydraulic loading rate of 0.5 m(3)/m(2)·d until clogging. Parameters related to the clogging of the wetland substrate before and after resting were examined and measured. The results showed that the resting operation could effectively alleviate bioclogging because the hydraulic conductivity and effective porosity were improved after 3, 7 and 10 days of resting. In the upper 0-10 cm layer, the hydraulic conductivity increased 2.0, 2.6 and 3.5 times, respectively, for the three resting periods. The reduction of the extracellular polymeric substance (EPS), biofilm decay and the consequential change in the biofilm structure are the main reasons that the resting operation relieved clogging. In addition, the observed and theoretical resting times (approximately 7 days) agreed well. The results provide a theoretical basis and technical support for solving clogging problems.

Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 µg/ml in milk.

A highly efficient site-specific integration strategy using combination of homologous recombination and the ΦC31 integrase.

The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications.

Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice.

Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.

The mesenchymal stem cells derived from transgenic mice carrying human coagulation factor VIII can correct phenotype in hemophilia A mice.

Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.

Bovine prolactin elevates hTF expression directed by a tissue-specific goat ß-casein promoter through prolactin receptor-mediated STAT5a activation.

Prolactin promotes the expression of exogenous human transferrin gene in the milk of transgenic mice. To elucidate this, a recombinant plasmid of bovine prolactin plus human transferrin vector was co-transfected into cultured murine mammary gland epithelial cells. Prolactin-receptor antagonist and shRNA corresponding to prolactin-receptor mRNA were added into the cell culture mixture to investigate the relations between prolactin-receptor and human transferrin expression after bovine prolactin inducement. Levels of human transferrin in the supernatants were increased under the presentation of bovine prolactin (from 1,076 ± 115 to 1,886 ± 114 pg/ml). With the treatment of prolactin-receptor antagonist or shRNA, human transferrin in cells was declined (1,886 ± 113 vs. 1,233 ± 85 pg/ml or 1,114 ± 75 pg/ml, respectively). An inverse correlation was found between the dosage of prolactin-receptor antagonist and expression level of human transferrin. Real-time qRT-PCR analysis showed that the relative level of signal transducer and activator of transcription 5a (STAT5a) transcript in transfected cells correlated with expression levels of human transferrin in the supernatant of the same cells. Bovine prolactin thus improved the expression of human transferrin through such a possible mechanism that bovine prolactin activated STAT5a transcription expression via combined with prolactin-receptor and suggest a potential utility of the bovine prolactin for efficient expression of valuable pharmaceutical proteins in mammary glands of transgenic animals.