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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.

To compare the regulation effects by different promoters on bovine prolactin gene expression in different cell lines, three recombinant bovine prolactin expression vectors were constructed using different promoters, i.e., CMV promoter, bovine prolactin gene promoter and goat beta-casein gene promoter, respectively named pCMV, pPRLP and pP1A3, which were transfected into two cell lines, mouse pituitary tumor cell strain (AtT20) and mouse mammary epithelial cell strain (HC11), respectively. RT-PCR and real-time RT-PCR were used to investigate the expression level of the above three vectors in both cell lines, pCMV vector was effectively expressed in both cell lines, pPRLP vector had a similar expression level to that of pCMV in both cell lines, pP1A3 was expressed in HC11 but not in AtT20. pP1A3 was tissue-specific to mammary gland, pPRLP was able to express with a significant level in pituitary and mammary glands, while its tissue-specific characteristics in other tissues need further investigation.

Large amounts of aberrantly spliced mRNA from the beta(654) allele was present in erythroid cells, which might impair the erythropoiesis. A therapeutic strategy for beta-thalassemia was explored by knocking down the aberrantly spliced mRNA of beta-globin. Lentiviral vector with siRNA fragment targets on the specific portion of beta(654)-globin aberrantly spliced pre-mRNA was constructed. In HeLa beta(654) cells, the siRNA vector could reduce approximately 60% of aberrantly spliced mRNA, which was assessed by RT-PCR and qRT-PCR. Furthermore, a disease model of beta(654) thalassemia mice with lentiviral-mediated siRNA was produced by subzonal injection (named Hbetai-Hbb(th-4)/Hbb(+) transgenic mice). Our results showed that the hemotological parameters were improved in Hbetai-Hbb(th-4)/Hbb(+) transgenic mice. This study provides a potential way for beta(654)-thalassemia therapy by knocking down the aberrantly spliced beta-globin mRNA, whilst supporting that the aberrantly spliced beta-globin mRNA may aggravate the disease.

Background: beta-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 beta-thalassemia and showed promise in clinical application. Using a human beta-globin transgenic mouse line in a beta-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome., Design and Methods: Human beta-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of beta(IVS-2-654)-thalassemia mice to generate a transgenic line. Human beta-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 beta-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 beta(IVS-2-654) mice carrying human beta-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 beta-globin transgenic mice in a beta(IVS-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 beta-thalassemia.

The beta-thalassemia is associated with abnormality in beta-globin gene, leading to imbalanced synthesis of alpha-/beta-globin chains. Consequently, the excessive free alpha-globin chains precipitate to the erythrocyte membrane, resulting in hemolytic anemia. We have explored post-transcriptional strategies aiming at alpha-globin reduction and beta-globin enrichment on beta(654) (Hbb(th-4)/Hbb(+)) mouse, carrying a human splicing-deficient beta-globin allele (Hbb(th-4)). Lentiviral vectors of short hairpin RNA (shRNA) targeting alpha-globin and/or antisense RNA facilitating beta-globin correct splicing were microinjected into beta(654) single-cell embryos. Three transgenic strains were generated, as alpha(i)-Hbb(th-4)/Hbb(+)(shRNA), beta(a)-Hbb(th-4)/Hbb(+)(antisense) and alpha(i)beta(a)-Hbb(th-4)/Hbb(+)(both shRNA and antisense). Without notable abnormalities, all the founders and their offsprings showed sustained amelioration of hematologic parameters, ineffective erythropoiesis and extramedullary hematopoiesis. Augmented effects appeared in alpha(i)beta(a)-Hbb(th-4)/Hbb(+), which correlated with a better-balanced alpha-/beta-globin mRNA level. Among the transgenic mice integrated with shRNA and antisense RNA, one homozygous mouse (Hbb(th-4)/Hbb(th-4)) had been viable, and the 3-week survival rate for heterozygotes (Hbb(th-4)/Hbb(+)) was 97%, compared with 45.4% for untreated. Our data have demonstrated the feasibility of techniques for beta-thalassemia therapy by balancing the synthesis of alpha-/beta-globin chains.

Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from nuclear transfer (NT) embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC (NT-ESC) lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage R1 donor cells were able to form full term developed pups, whereas those from late passage R1 ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-R1-ESC lines that were developed from NT blastocyst of late passage R1 ESC donors. However, these NT-R1-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.

Objective: To investigate the expression of human specific proteins in liver tissue obtained from goats engrafted with human hematopoietic stem cells (hHSC)., Methods: hHSCs derived from cord blood were transplanted into fetal goats for production of chimerism. Liver specimens were obtained from four 10-month-old chimeric goats and examined for their reactivity with monoclonal antibodies against human antigens: proliferating cell nuclear antigen (PCNA) and hepatocyte specific antigen (HSA). The presence of human HSA positive cells in the goat liver tissue was determined by fluorescence assisted cell sorting (FACS). The expression of human hepatocyte nuclear factor (hHNF-3beta) and human serum albumin (hALB) mRNAs was determined by RT-PCR. Meanwhile, FISH experiment was also performed to detect the human cells in the chimeric livers with the probe of human p17H8., Results: Successful engraftment was confirmed by the detection of human blood cell chimerism in the circulation of the transplanted goats. Human PCNA and HSA were found in liver specimens of transplanted goats but not of normals. FACS analysis showed the presence of human HSA positive cells in the liver of chimeric goats. RT-PCR results demonstrated that hHNF-3beta and hALB mRNAs were specifically expressed in liver tissues of chimeric goats. FISH experiment showed two positive hybridized signals in some liver cells of the chimeric goats, indicating that human liver-like cells were present there., Conclusion: Goats engrafted with hHSC are capable to produce chimeric livers. Growth and propagation of human cells in the liver tissue of such transplanted goats were possible. Particularly, the human liver-like cells in chimeric goat livers had transcriptional activity specific to human hepatocytes.

Objective: To establish a human/goat hematopoietic stem cell (HSC) xenogeneic transplant model and to probe the engraftment, expansion and differentiation of human HSC in vivo., Methods: Human HSCs were isolated from human umbilical cord blood and 1 x 10(5) human HSCs were in utero transplanted into 50 fetal goats at the 55 - 65 the gestation days. The engraftment, expansion and differentiation of human HSCs in goats were determined by FACS analysis, PCR and PCR-Southern blot hybridization at various intervals after birth., Results: Hematopoietic chimerism occurred certainly in 35 of 39 live-born recipients. On average, The proportion of human hematopoietic cells in goat blood was 1% approximately 3% and remained phenotypically stable for at least 10 months. The human hematopoietic cells circulated in goat blood expressed CD34, CD14, CD20 and glycophorin A (GPA) but did not express CD3, CD4, CD7, CD8 and CD56 or expressed them at a very low level., Conclusion: The number of human HSC can be effectively expanded 1 000 - 10 000 fold. Human HSCs in goats undergo a limited differentiation. Our human/goat HSC xenogeneic model provides a useful way for the investigation of HSC transplantation, expansion and differentiation in vivo.