Shuzhen Huang

Multiorgan engraftment and differentiation of human cord blood CD34+ Lin- cells in goats assessed by gene expression profiling.

To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+Lin− cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45–55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2–36% of all cells examined). We identified human β2 microglobulin-positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte-specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions.

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer

BackgroundThe interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT.ResultsWe investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog).ConclusionBoth histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

A ϕC31 integrase‐mediated integration hotspot in favor of transgene expression exists in the bovine genome

ϕC31 integrase, a site‐specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were discovered using half‐nested inverse PCR from cow fibroblasts. The genomic locations of these two pseudo attP sites were identified by direct sequencing and a BLAST search, and it was confirmed that they reside at positions 4q31 and 10q35 by fluorescence in situ hybridization analysis. Subsequently, the distinct integration frequencies of the four pseudo attP sites were examined. The BF4 site was identified as a hotspot where site‐specific integration occurred in most of the cell clones examined, accounting for 74% (42/57) of the integration; much more than the integration frequency for BF10 (7%; 4/57), BpsF1 (7%; 4/57) and BpsM1 (0/57). Interestingly, similar to other hotspots identified in the human and mouse genomes, in which transgenes integrated at hotspots result in high expression, the GFP gene integrated at hotspot BF4 was expressed at high levels in cow fibroblasts, as confirmed by fluorescence microscopy and FACS analysis. Furthermore, ELISA showed that the expression level of the GFP gene integrated at the BF4 site averaged ∼ 328 μg·mg−1, which is more than twofold higher than that integrated at the BF10 site. This study suggests that somatic cells carrying a desired gene integrated at the BF4 site can be used as nuclear donors to generate valuable transgenic animals by nuclear transfer.

Correction of β654-thalassaemia mice using direct intravenous injection of siRNA and antisense RNA vectors

Although the therapeutic efficacy of β654-thalassaemia treatment using a combination of RNAi and antisense RNA to balance the synthesis of α- and β-globin chains has been demonstrated previously, and the safety of lentiviral delivery remains unclear. Herein, we used the same β654-thalassaemia mouse model to develop a therapy involving direct delivery of siRNA and antisense RNA plasmids via intravenous injection to simultaneously knock down α-globin transcript levels and restore correct β-globin splicing. The amount of α-globin mRNAs in siRNA-treated MEL cells decreased significantly, and the properly spliced β-globin mRNA was restored in HeLaβ654 cells transfected with pcDNA-antisense plasmid. Furthermore, treatment of β654-thalassaemic mice with siRNA and antisense RNA plasmids resulted in significant reduction of poikilocytosis and reticulocyte counts in blood samples, decreased nucleated cell populations in bone marrow, and reduced intrasinusoidal extramedullary haematopoiesis loci and iron accumulation in liver. RT-PCR analysis revealed that treatment resulted in down-regulation of α-globin mRNA synthesis by ~50% along with an increase in the presence of normally spliced β-globin transcripts, indicating that the phenotypic changes observed in β654-thalassaemic mice following treatment resulted from restoration of the balance of α/β-globin biosynthesis.

Aberrant expression of imprinted genes and their regulatory network in cloned cattle

Domesticated animals cloned by somatic cell nuclear transfer (SCNT) generally have poor developmental competency, with many developmental abnormalities attributed to incomplete reprogramming of the nuclear genome and abnormal expression of genes important for regulation of growth and development. To investigate the molecular mechanism leading to the abnormalities of cloned animals, pathologic and histologic analyses were conducted on seven cloned cattle that were oversized at birth and had cardiac and pulmonary abnormalities. Quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis of four imprinted genes IGF2, IGF2R, H19, and GRB10, as well as genes from related regulatory networks, were performed in liver, lung, kidney, and muscle to investigate disruption of expression. Expression of IGFBP2 was not detected in morphologically normal cloned cattle, but was detected in the liver, lung, kidney, and thymus of abnormal calves. Expression levels of IGF1 and imprinted genes IGF2 and H19 were substantially higher in these organs of abnormal cattle. In contrast, expression levels of GRB10, CTSD, and TRPV2 were substantially lower in abnormal cattle. Transcript abundance of IGFBP6 was higher in kidney, but lower in liver and lung. In conclusion, we inferred that altered expression of imprinted and related genes may be closely related to increased birth weight and pathologic changes in abnormal cloned cattle.

A profile of native integration sites used by φC31 integrase in the bovine genome.

The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 (DLC1) gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed. We conclude that this site-specific integrase system has great potential for applied modifications of the bovine genome.

Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning.

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.

A novel transgenic mouse model produced from lentiviral germline integration for the study of β-thalassemia gene therapy

Lentiviral-mediated gene therapy has been successfully applied in the treatment of β-thalassemia. In this study transgenic mice with stable expression of a lentivirus carrying the human β-globin gene were obtained. These animals provide a useful model to investigate the stable effect of gene therapy in β-thalassemia. Background β-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of β-thalassemia and showed promise in clinical application. Using a human β-globin transgenic mouse line in a β-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. Design and Methods Human β-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of βIVS-2-654-thalassemia mice to generate a transgenic line. Human β-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. Results Transgenic mice with stable expression of the lentivirus carrying human β-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the βIVS-2-654 mice carrying human β-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. Conclusions The generation of human β-globin transgenic mice in a βIVS-2-654-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in β-thalassemia.

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.

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55+/-1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.