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.

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.

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.

Genome-wide hypermethylation is closely associated with abnormal expression of genes involved in neural development in induced pluripotent stem cells derived from a Down syndrome mouse model.

Mental retardation is the main clinical manifestation of Down syndrome (DS), and neural abnormalities occur during the early embryonic period and continue throughout life. Tc1, a model mouse for DS, carries the majority part of the human chromosome 21 and has multiple neuropathy phenotypes similar to patients with DS. To explore the mechanism of early neural abnormalities of Tc1 mouse, induced pluripotent stem (iPS) cells from Tc1 mice were obtained, and genome-wide gene expression and methylation analysis were performed for Tc1 and wild-type iPS cells. Our results showed hypermethylation profiles for Tc1 iPS cells, and the abnormal genes were shown to be related to neurodevelopment and distributed on multiple chromosomes. In addition, important genes involved in neurogenesis and neurodevelopment were shown to be downregulated in Tc1 iPS cells. In short, our study indicated that genome-wide hypermethylation leads to the disordered expression of genes associated with neurodevelopment in Tc1 mice during early development. Overall, our work provided a useful reference for the study of the molecular mechanism of nervous system abnormalities in DS.

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.

A mediator of phosphorylated Smad2/3, evodiamine, in the reversion of TAF-induced EMT in normal colonic epithelial cells.

Purpose Transdifferentiation exists within stromal cells in the tumour microenvironment. Transforming growth factor-ß (TGF-ß) secreted by tumour-associated fibroblasts (TAFs) affects the differentiation states of epithelial cells, including epithelial-mesenchymal transition (EMT). Evodiamine, a natural drug, can regulate differentiation. However, the specific effects and relative mechanisms of evodiamine remain unknown. Design We used four models to observe the influence of TAF-like CCD-18Co cells on the colon epithelial cell line HCoEpiC: the 3D- and 2D-mono-culture system, Transwell and direct co-culture model. Additionally, we established conditioned medium from CCD-18Co cells. The TGF-ß pathway inhibitor LY364947 and evodiamine were added. Morphological changes and classical EMT markers were observed and detected using phase contrast microscopy and immunofluorescence. Cell migration was measured by the wound-healing assay. Western blotting was performed to detect the TGF-ß/Smad signalling pathway. Results CCD-18Co cells induced EMT-like changes in the 2D- and 3D-cultured epithelial cell line HCoEpiC, accompanied by high expression of ZEB1 and Snail and the enhancement of migration. Moreover, CCD-18Co-derived conditioned medium caused dysfunction of TGF-ß/Smad signalling in EMT. Evodiamine inhibited these EMT-like HCoEpiC and their migration. Additionally, evodiamine down-regulated the expression of ZEB1/Snail and up-regulated the expression of phosphorylated Smad2/3 (pSmad2/3). Evodiamine also increased the ratios of pSmad2/Smad2 and pSmad3/Smad3. Conclusion Based on our observations, evodiamine can reverse the TAF-induced EMT-like phenotype in colon epithelial cells, which may be associated with its mediation of phosphorylated Smad2 and Smad3 expression.

Treatment of ß654 -thalassaemia by TALENs in a mouse model.

OBJECTIVES: This study explored whether TALENs-mediated non-homologous end joining (NHEJ) targeting the mutation site can correct the aberrant ß-globin RNA splicing, and ameliorate the ß-thalassaemia phenotype in ß654 mice. MATERIAL AND METHODS: TALENs vectors targeted to the human ß-globin gene (HBB) IVS2-654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs+ /ß654 mice. The gene editing and off-target effects were analysed by sequencing analysis. ß-globin expression was identified by RT-PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs+ /ß654 mice. RESULTS: Sequencing analysis revealed that the HBB IVS2-654C >T point mutation was deleted in over 50% of the TALENs+ /ß654 mice tested, and off-target effects were not detected. RT-PCR and Western blot analysis confirmed the expression of normal ß-globin in TALENs+ /ß654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs+ /ß654 mice. CONCLUSION: These results suggest effective treatment of the anaemia phenotype in TALENs+ /ß654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of ß654 -thalassaemia in the future.

A Herpes Simplex Virus Thymidine Kinase-Induced Mouse Model of Hepatocellular Carcinoma Associated with Up-Regulated Immune-Inflammatory-Related Signals.

Inflammation and fibrosis in human liver are often precursors to hepatocellular carcinoma (HCC), yet none of them is easily modeled in animals. We previously generated transgenic mice with hepatocyte-specific expressed herpes simplex virus thymidine kinase (HSV-tk). These mice would develop hepatitis with the administration of ganciclovir (GCV)(Zhang, 2005 #1). However, our HSV-tk transgenic mice developed hepatitis and HCC tumor as early as six months of age even without GCV administration. We analyzed the transcriptome of the HSV-tk HCC tumor and hepatitis tissue using microarray analysis to investigate the possible causes of HCC. Gene Ontology (GO) enrichment analysis showed that the up-regulated genes in the HCC tissue mainly include the immune-inflammatory and cell cycle genes. The down-regulated genes in HCC tumors are mainly concentrated in the regions related to lipid metabolism. Gene set enrichment analysis (GSEA) showed that immune-inflammatory-related signals in the HSV-tk mice are up-regulated compared to those in Notch mice. Our study suggests that the immune system and inflammation play an important role in HCC development in HSV-tk mice. Specifically, increased expression of immune-inflammatory-related genes is characteristic of HSV-tk mice and that inflammation-induced cell cycle activation maybe a precursory step to cancer. The HSV-tk mouse provides a suitable model for the study of the relationship between immune-inflammation and HCC, and their underlying mechanism for the development of therapeutic application in the future.

Effect of evodiamine and berberine on the interaction between DNMTs and target microRNAs during malignant transformation of the colon by TGF-ß1.

The tissue microenvironment functions as a crucial player in carcinogenesis, and transforming growth factor-ß1 (TGF-ß1) within the microenvironment stimulates the formation of neoplasms. Using an in vitro model of malignancy induced by TGF-ß1, we assessed the effect of evodiamine and berberine on the interaction between DNA methyltransferases (DNMTs) and target microRNAs (miRNAs) in the model. Colon tissues from neonatal rats 7 days of age were cultured and malignancy was induced by TGF-ß1 in vitro for 48 h, and then the tissues were respectively treated with evodiamine and berberine for 24 h. Morphological alteration of tissues was observed by an inverted microscope, histological structures were observed using hematoxylin and eosin staining, and the expression levels of DNMTs and targeted miRNAs screened by bioinformatics software combined with Gene chip analysis in our previous study were detected by immunohistochemistry and quantified by real-time PCR. Twenty-four hours after treatment with TGF-ß1, expression levels of DNMT1, DNMT3A, DNMT3B and miR-152 (target DNMT1), miR-429 (target DNMT3A) and miR-29a (target DNMT3A/3B) were markedly decreased; however, after 48 h, the expression levels of DNMT1 and DNMT3A were significantly increased, but their target miRNAs were still decreased. After treatment with a DNMT inhibitor (5-Aza-dC), expression levels of the miRNAs were increased to a larger extent, but did not reach normal levels. After treatment with berberine and evodiamine for 24 h, respectively, increased expression of DNMT1, DNMT3A, DNMT3B and miR-152, miR-429, miR-29a was noted. In conclusion, the results of the present study suggest that miRNAs can also be post-transcriptionally regulated by their corresponding DNMTs and that berberine and evodiamine regulate the expression of these genes, which provides early epigenetic evidence for the prevention and therapy of colorectal cancer.

[Corrigendum] Analysis of different components in the peritumoral tissue microenvironment of colorectal cancer: A potential prospect in tumorigenesis.

Owing to an oversight during the proof checking stage, the above article has been published with the incorrect author listed for correspondence. The first author, Chao Huang, is listed as the corresponding author, although he was only intended to have been temporarily assigned to handle queries during the pre-press stages of the publication. The correct corresponding author should have been listed as Professor Bin Wen (also at the Spleen­Stomach Institute, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510000, P.R. China). The email address for Professor Wen is: wenbin@gzucm.edu.cn. We sincerely apologize for this mistake, and regret any inconvenience this mistake has caused [the original article was published in the Molecular Medicine Reports 14: 2555-2565, 2016; DOI: 10.3892/mmr.2016.5584].

Expression of DNA methyltransferases and target microRNAs in human tissue samples related to sporadic colorectal cancer.

Tissue microenvironment functions as a pivotal mediator in colorectal carcinogenesis, and its alteration can cause some important cellular responses including epigenetic events. The present study examined histologically altered tissue structure, DNA methyltransferases (DNMTs) and their corresponding expression of target microRNAs (miRNA). Tissues resected by surgery were from primary colorectal carcinoma. These samples were from three locations: and were ≥10, 5 and ≤2 cm away from the proximal lesion of colon cancer, and marked as no. 1, no. 2 and no. 3, respectively. Histological alteration was assessed by H&E staining, expression of DNMT1, DNMT3A, and DNMT3B was detected by immunohistochemistry and western blotting, microarray chip was used to screen distinguishable miRNAs and miRNAs targeting DNMTs whose validation assay was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Our results revealed that normal crypt structure was shown in no. 1, while many aberrant crypt foci appeared in no. 3. Significant upregulation of DNMT1, DNMT3A, and DNMT3B expression was found in para-carcinoma tissues, compared with the histopathologically unchanged tissues (P<0.05), furthermore, distinguishable expression profiling was observed of target miRNAs in tissues with different distance. Our results provide additional insights for future research of colorectal carcinogenesis by introducing the tissue microenvironment.

Analysis of different components in the peritumoral tissue microenvironment of colorectal cancer: A potential prospect in tumorigenesis.

The present study aimed to observe the varying expression of biomarkers in the microenvironment adjacent to colorectal cancer lesions to provide additional insight into the functions of microenvironment components in carcinogenesis and present a novel or improved indicator for early diagnosis of cancer. A total of 144 human samples from three different locations in 48 patients were collected, these locations were 10, 5 and 2 cm from the colorectal cancer lesion, respectively. The biomarkers analyzed included E­cadherin, cytokeratin 18 (CK18), hyaluronidase­1 (Hyal­1), collagen type I (Col­I), Crumbs3 (CRB3), vimentin, proteinase activated receptor 3 (PAR­3), α­smooth muscle actin (α­SMA), cyclin D1 (CD1) and cluster of differentiation (CD)133. In addition, crypt architecture was observed. Related functional analysis of proteins was performed using hierarchical index cluster analysis. More severe destroyed crypt architecture closer to the cancer lesions was observed compared with the 10 cm sites, with certain crypts degraded entirely. Expression levels of E­cadherin, CK18, CRB3 and PAR­3 were lower in 2 cm sites compared with the 10 cm sites (all P<0.001), while the expression levels of the other biomarkers in the 2 cm sites were increased compared with 10 cm sites (all P<0.0001). Notably, the expression of CK18 in 2 cm sites was higher than in the 5 cm site (P<0.0001), which was different from the expression of E­cadherin, CRB3 and PAR­3. The expression levels of Hyal­1 and Col­I at the 2 cm sites were lower than that of the 5 cm sites (P>0.05 and P=0.0001, respectively), while the expression of vimentin, α­SMA, CD1 and CD133 were not. Hyal­1 and Col­I may be independently important in cancer initiation in the tumor microenvironment. The results of the present study suggest that the biomarkers in the tissue microenvironment are associated with early tumorigenesis and may contribute to the development of carcinomas. These observations may be useful for early diagnosis of colorectal cancer.

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.

Induction of protection against foot-and-mouth disease virus in cell culture and transgenic suckling mice by miRNA targeting integrin αv receptor.

Foot-and-mouth disease virus (FMDV) is an RNA virus that causes a highly contagious disease in domestic and wild cloven-hoofed animals. Although vaccination has been used to protect animals against FMDV, there are shortcomings in the efficacy of the available vaccines. RNA interference (RNAi) is triggered by small RNA molecules, including short interfering RNAs and microRNAs (miRNAs), and the use of RNAi-based methods have demonstrated promise as an alternative method of controlling the transmission of FMDV. However, the method of delivery, short duration of siRNA and miRNA in vivo, and the genetic variability of FMDV confound the use of RNAi-based strategies for FMDV control. FMDV has been shown to exploit host-cell integrins as cell-surface receptors to initiate infection. We selected the gene for the integrin αv subunit as an RNAi target, and constructed three αv-specific miRNA expression plasmids. The effects of these miRNAs on FMDV infection were examined in PK-15 cells and transgenic suckling mice. In PK-15 cells, the expression of the αv-specific miRNAs significantly inhibited the expression of integrin αv receptor and decreased FMDV infection. The transgenic mice were generated by integrating the αv-specific miRNA expression cassette using pronuclear microinjection. When challenged with a dose of FMDV ten times greater than the LD50, the survival rate of transgenic suckling mice was approximately six-fold higher than that of their non-transgenic littermates, indicating that the interference of the miRNAs significantly reduced FMDV infection in the transgenic mice. This is the first report of limiting FMDV attachment to cellular receptors using miRNA-mediated gene knock down of cell-surface receptors to significantly reduce FMDV infection in cell culture and transgenic suckling mice.

[Isolation and gene modification of amniotic fluid derived progenitor cells].

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.

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.

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.

Engraftment of genetically modified human amniotic fluid-derived progenitor cells to produce coagulation factor IX after in utero transplantation in mice.

Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B.

A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation.

BACKGROUND: Major improvements have been made progressively on human immunodeficiency virus (HIV)-1 based lentiviral vectors to minimize the probability of replication-competent lentivirus formation. This includes the deletion of U3 promoter and the use of packaging cells, which has increased their potential for use in gene therapy and other in vivo applications. However, the risk of forming replication-competent lentiviruses remains. METHODS: We investigated the use of Cre-loxP mediation with the insertion of the transgene-expressing cassette in ΔU3 to remove additional parts of the HIV-1 backbone upon cre expression, after integration. This, leads to deletion of the packaging signal, primer binding site and Rev response element, including cre itself. RESULTS: This approach left a split truncated form of long terminal repeat flanked by a loxP and a transgene-expressing cassette in the genome, which made replication-competent lentivirus formation almost impossible. This self-deletion vector could stably express transgenes both in cell lines and transgenic mice with only modest losses of viral titer. The maximum size of the inserts was approximately 3 kb, which was sufficient for most transgenic applications. Moreover, the addition of some enhancer blocking agents downstream of the transgene could reduce the probability of transcriptional read-through in transfected 293T cells. CONCLUSIONS: Our approach could improve the biosafety of lentiviral vectors, thus improving their potential application for use in clinical trials and other in vivo applications.