Hypoxia enhances the therapeutic potential of superparamagnetic iron oxide-labeled adipose-derived stem cells for myocardial infarction.

Adipose-derived stem cells (ASCs) induce therapeutic angiogenesis due to pro-angiogenic cytokines secretion. Superparamagnetic iron oxide (SPIO) nanoparticles are critical for magnetic resonance (MR) tracking of implanted cells. Hypoxia is a powerful stimulus for angiogenic activity of ASCs. In this study, we investigated whether therapeutic potency could be enhanced by implantation of hypoxia-preconditioned SPIO-labeled ASCs (SPIOASCs) into the infarcted myocardium. ASCs and SPIOASCs were cultured under 2% O2 (hypoxia) or 95% air (normoxia). Cells were intramyocardially injected into the infarcted myocardium after 48-h culture. We found that hypoxia culture increased the mRNA expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in ASCs and SPIOASCs. The VEGF protein in the conditioned medium was significantly higher in hypoxic ASCs and SPIOASCs than in normoxic ASCs and SPIOASCs. The capillary density and left ventricular contractile function in the infarcted myocardium were significantly higher 4 weeks after implantation with hypoxic ASCs and SPIOASCs than with normoxic ASCs and SPIOASCs. Improvement in the capillary density and left ventricle function didn't differ between hypoxic ASCs-transplanted rats and hypoxic SPIOASCs-transplanted rats. Hypoxic culture enhanced the angiogenic efficiency of ASCs. It was concluded that implantation of hypoxic ASCs or SPIOASCs promotes therapeutic angiogenesis and cardiac function recovery in the infarcted myocardium. SPIO labeling does not impact the beneficial effect of hypoxic ASCs.

Hypoxia Enhances the Therapeutic Potential of Superparamagnetic Iron Oxide-labeled Adipose-derived Stem Cells for Myocardial Infarction / 华中科技大学学报（医学）（英德文版）

Adipose-derived stem cells (ASCs) induce therapeutic angiogenesis due to pro-angiogenic cytokines secretion.Superparamagnetic iron oxide (SPIO) nanoparticles are critical for magnetic resonance (MR) tracking of implanted cells.Hypoxia is a powerful stimulus for angiogenic activity of ASCs.In this study,we investigated whether therapeutic potency could be enhanced by implantation of hypoxia-preconditioned SPIO-labeled ASCs (SPIOASCs) into the infarcted myocardium.ASCs and SPIOASCs were cultured under 2％ O2 (hypoxia) or 95％ air (normoxia).Cells were intramyocardially injected into the infarcted myocardium after 48-h culture.We found that hypoxia culture increased the mRNA expression of hypoxia-inducible factor-1 alpha (HIF-lαt) and vascular endothelial growth factor (VEGF) in ASCs and SPIOASCs.The VEGF protein in the conditioned medium was significantly higher in hypoxic ASCs and SPIOASCs than in normoxic ASCs and SPIOASCs.The capillary density and left ventricular contractile function in the infarcted myocardium were significantly higher 4 weeks after implantation with hypoxic ASCs and SPIOASCs than with normoxic ASCs and SPIOASCs.Improvement in the capillary density and left ventricle function didn't differ between hypoxic ASCs-transplanted rats and hypoxic SPIOASCs-transplanted rats.Hypoxic culture enhanced the angiogenic efficiency of ASCs.It was concluded that implantation of hypoxic ASCs or SPIOASCs promotes therapeutic angiogenesis and cardiac function recovery in the infarcted myocardium.SPIO labeling does not impact the beneficial effect of hypoxic ASCs.

Malignant transformation of 293 cells induced by ectopic expression of human Nanog.

Tumor development has long been known to resemble abnormal embryogenesis. The ESC self-renewal gene NANOG is purportedly expressed in some epithelial cancer cells and solid tumors, but a casual role in tumor development has remained unclear. In order to more comprehensively elucidate the relationship between human Nanog and tumorigenesis, the hNanog was ectopically expressed in the 293 cell line to investigate its potential for malignant transformation of cells both in vitro and in vivo. Here we provide compelling evidence that the overexpression of hNanog resulted in increased cell proliferation, anchor-independent growth in soft agar, and formation of tumors after subcutaneous injection of athymic nude mice. Pathologic analysis revealed that these tumors were poorly differentiated. In analysis of the underlying molecular mechanism, two proteins, FAK and Ezrin, were identified to be upregulated in the hNanog expressing 293 cells. Our results demonstrate that hNanog is a potent human oncogene and has the ability to induce cellular transformation of human cells.

[Multipotential differentiation and potential applications of adipose-derived stem cells].

Adipose tissue contains a population of multipotent cells called adipose-derived stem cells (ADSCs). With the similar properties of marrow-derived mesenchymal stem cells, ADSCs have the ability to differentiate differentiate towards adipogenic, osteogenic, chondrogenic, myogenic, endothelial, hematopoietic, hepatic, islet, and neurogenic cell lineages. As adipose tissue in harvested in large amounts with minimal morbidity, it can be widely used in tissue engineering, organ repair and gene therapy. This paper focused on the plasticity of ADSCs and reviewed the new advances of this field. Finally, the problems and prospect for application was also discussed.

[The targeting study of human serum albumin gene].

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

[Accelerated somatic cell senescence and changes in p16INK4a expression after exogenous DNA transfection].

During the transfection of mouse embryonic fibroblasts (MEF), we found these cells became senescent, appearing enlarged with hollow cytoplasm and multinucleated. The telomere lengths of these senescent MEFs were significantly shorter than primary untransfected MEFs. In senescent MEF cells, DNA methylation of p16INK4a 5'-regulatory region showed gradual reduction as cells aged. RT-PCR and Northern blot demonstrated that the expression of p16INK4a in transfected senescent cells was 12-16 times more than primary cells. The senescent transfected MEF cells as donors of nuclei could support the early development of cloned embryos after nuclear transfer.

[The synthesis and function analysis of omega-3 fatty acid desaturase gene from Caenorhabditis briggssae].

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.

[Synthesis of total sFat-1 gene by PCR-restrict enzyme ligation method].

Gene synthesis is very important in life science research, and it becomes a technique in common use. It is difficult for long gene synthesis, because the mismatches and mutations of DNA sequence in nucleotide fragments assembling. This study established a new method for long gene synthesis, which was referred to as PCR-restrict enzyme ligation method. With this method, a omega-3 fatty acid desaturase gene, sFat-1, from Caenorhabditis briggssae, was successfully assembled from 27 synthesized nucleotide fragments (60 ~ 68 bp for each fragment ) following 3 rounds of PCR (7 reactions) and 2 rounds of restrict enzyme ligation (3 reactions). This shows that the PCR-restrict enzyme ligation method is an effective method for long gene synthesis.

[Characteristics and Influencing factors of Sperm Interaction with Exogenous DNA].

Exogenous DNA localization and the frequency of spermatozoa carrying exogenous DNA after sperm/DNA co-culture are key to a successful sperm mediated-gene transfer (SMGT). In the study, the characteristics and influencing factors of exogenous DNA uptake by spermatozoa were tested using digoxigenin (DIG) labeled DNA as trace. Results showed that goat spermatozoa could spontaneously take up exogenous DNA. The exogenous DNA was initially bound to the outer sperm membrane at postacrosomal region; subsequently party of the bound DNA was internalized into nucleus. There were considerable differences in the capability of spermatozoa from different donors to bind and internalize exogenous DNA. In 35 samples, binding rates (before DNase I digestion) and internalization rates (the positive rate after DNase I digestion) varied between 4.6%-62.4% and 2.1%-53.8%, respectively. For the spermatozoa from the same goat, the binding and internalization capacities were mostly inhibited by the seminal fluid. Compared to ejaculate sperm, the binding rate and internalization rate were increased three and five times in washed sperm cells, respectively. At the same time, capacitated spermatozoa also had lower exogenous DNA uptake (P<0.01). Dead spermatozoa did not complete the internalization process. The highest positive rate (before DNase I digestion) was found in membrane-broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors. These results suggest that selection of appropriate sperm donors and optimization of sperm processing procedures are the key steps for successful SMGT.

[The construction and characterization of human pro-urokinase mutant].

It is very easy for the pro-UK to lose it's biological activity because of the digestion of pro-UK by the thrombin or the inhibition of pro-UK by the PAI-I. So three pro-UK mutant (pro-UK) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg156 in pro-UK was mutated into His156, and named as pro-UKM1; PAI binding sites Arg178, Arg179, Arg181 were mutated into Lys178, Lys179, His181, named as pro-UKM2; The mutant containing His156, Lys178, Lys179, His181 as pro-UKM3. Three mutants were expressed in CHO cells respectively and analyzed with SDS-PAGE fibrin plate assay and western blot. The results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK2 could resist against PAI inhibition; while pro-UK3 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK3 can be a new medicine of dissolving thrombus.

[High-efficient gene targeting of goat mammary epithelium cell by the multi-selection mechanism].

Gene targeting is a more powerful method to produce mammary gland bioreactor and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe an efficient and reproducible gene targeting in goat mammary epithelium cell to place the GFP and neo at the beta-casein locus. The transgenic goat would be produced by nuclear transfer. To construct the gene targeting vector pGBC-GFP-neo, the milk goat beta-casein genomic DNA sequence for homologous arms was cloned first. The left arm was 2.1 kb fragment including goat beta-casein gene exon1 and part of exon 2, and the right arm was 5.1 kb fragment including beta-casein gene from exon 7 to 3'-flanking sequence. The bacterial neomycin (neo) gene as positive selection marker gene, with the promoter-trap GFP, was placed between two loxPs. The thymidine kinase (tk) as negative selection marker gene was just outside the right or left arms. Goat mammary epithelium cells were cultured to sub-confluence about 90% and transfected with linear pGBC-GFP-neo using Lipefectamin-2000. These transfected cells were cultured in collagen-coated 96-wellplate for 24 h without selection, then added the drug G418(600 microg/mL) and GANC(2 micromol/L). After nine days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate; 51 clones were selected; 17 clones were tested by GFP expression using promoter-trap strategy; only four clones grow well. After PCR confirmation the four targeting cell clones homologous recombination were used as the donor cell for nuclear transfer. 59.5% cloned embryos could develop up. Some could develop to morula or blastocyst in vitro.

[Study on a human tissue-type plasminogen activator mutant cDNA knocked-in the beta-casein gene site of murine ES cells].

A murine beta-casein gene targeting vector was constructed using the cloned genomic sequence. The short arm was 2.7 kb including mouse beta-casein gene 5' flanking sequence, exon1, intron1 and partial exon2. The long arm is a 3.4 kb fragment including partial intron2, exon3 approximately 7, intron3 approximately 6 and partial intron7. The human t-PA mutant cDNA was subcloned in the exon2 and fused with the mice beta-casein signal peptide sequence. The positive selective marker neo was placed in the middle of intron2. A tk negative selective marker was just outside the short arm. TC-1 ES cells were cultured and amplified on G418 resistant feeder layer. The linearized targeting construct DNAs of 45 microg were introduced into 2 x 10(7) ES cells by electroporation. Totally 192 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days. The colonies were amplified and subjected to genomic DNA preparation. The genomic DNAs were digested with EcoR I and used for Southern blot analysis. A probe inside the 5' homologous arm was used for hybridization. A 9.8 kb band was found in wild type, but the band was shift down from 9.8 kb to 6.6 kb in the beta-casein gene targeted allele because a new EcoR I site was introduced into the exon2 along with the human t-PA mutant gene. There were 9.8 kb and 6.6 kb bands in targeted ES cells. One clone of targeted ES cells with correct homologous recombination events was obtained among 78 analyzed clones. It lays foundation for gene targeted mice making.

[Nuclear reprogramming of somatic nuclear transfer embryos].

Although the cloned animals have been successfully generated in a number of mammalian species, there are still many problems about this technology. The developmental aberrancies include a high rate of abortion during early gestation and high rate of perinatal death. The main cause of these problems may be attributed to the epigenetic reprogramming of somatic donor genome. During mammalian embryonic development, DNA methylation is an essential process in the regulation of transcription. There are many aberrant methylation in various genomic regions of cloned embryos. The gene imprinting of cloned embryos are also abnormal.

A human t-PA mutant cDNA cassette knocked in the murine fgfr-4 locus targeting for mammary gland expression.

The expression of foreign gene in transgenic animals produced by pronuclear microinjection is often confounded by the position effects caused by not only the nature of chromosomal integration site but also the number and arrangement of multiple transgene copies. Gene targeting provides a new way to overcome these inhibitions by introducing single-copy transgene into a chosen site. The choice of a good chromosomal site will favor transgene expression in a predictable fashion. In this study, we tested a new site (fgfr-4) for foreign gene integration and expression. A t-PA mutant (t-PAm) expression cassette under bovine alphas1-casein regulatory sequences was efficiently knocked-in fgfr-4 site through homologous recombination. The t-PAm was expressed in the milk of all targeted mice. Our experiment indicates that the fgfr-4 may be a candidate site for knocking foreign gene to make transgenic animals.

[The ht-PAm cDNA knock-in the goat beta-casein gene locus].

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.

[The transgenic mice specifically express Cre recombinase in central nerve system].

For specific expressing Cre recombinase in central nerve system (CNS), a transgenic construct (pGFAP-Cre-hGH), containing the beta-globin insulators, 1.8 kb of glial fibrillary acidic protein gene (GFAP) 5' end regulation region, Cre gene and polyA of human growth hormone gene (hGH) was generated, in which the 5' end regulation region of GFAP was isolated from a 129sv mouse genomic DNA library with PCR-screening. 7.6 kb of pGFAP-Cre-hGH DNA fragment was introduced into 191 fertilized eggs by microinjection. 176 injected eggs were implanted into the oviducts of eight female mice respectively, from which 25 offspring were obtained. Seven mice carry the Cre genes by the identification of PCR and Southern blotting, and the integration efficiency is 28%. The GFAP-Cre transgenic mice were crossed with ROSA26 mice whose genomic DNA is integrated by LoxP sites and LacZ expression frame to check the activity and the tissue-specific expression of Cre recombinase and recombination with its mediation between two LoxP sites. The results of LacZ dying showed that the Cre recombinase was expressed only in CNS and successfully mediated the recombination between the LoxP sites in vivo.

[Production of mammary gland bioreactor by gene targeting of somatic cells].

Producing mammary gland bioreactor showed great advantage over many years, but the level of transgenic expression was low in transgenic animals and the diversity was more great because of the position effect of transgene and the artificial recombination of the gene elements. Gene targeting based on the principle of gene homologous recombination had been studied and applied, because the transgene could be integrated precisely in the chromosome. This review summary the current status of producing mammary gland bioreactor by the technology of gene targeting and nuclear transfer using the somatic cell lines. These aspects were discussed, including the characteristic and difficulties of gene targeting, the strategies to improve the efficiency of gene targeting, the different features of between the strategy of promoter-trap and the Cre-LoxP system, etc; for the others, how to select the cell lines with the different strategies of gene targeting, how to raise the times of cell lines that was cultured after the gene targeting. Somatic cell nuclear transfer offers new and exciting opportunities in the areas of the gene targeting. However, the field as a whole is still difficult and complex. In this paper, we described recent advances and novel approaches, which resulted in progress during the last year. Key problems hindering further progress are addressed, for example, how to increase the efficiency of nuclear transfer. With the technology of gene targeting and nuclear transfer, it should provide a general way to produce specific genetic changes in several mammalian species. We are clearly at the dawn of a new era in mammalian genetic technology.

[Murine beta-casein gene sequences direct a human tissue plasminogen activator variant expressed in the milk of transgenic mice].

To investigate the ability of our cloned murine beta-casein locus to direct the exogenous gene expression in the milk of transgenic mice, the human t-PA variant mammary gland expression vector under the control of murine beta-casein gene regulatory elements was constructed, in which the human t-PA variant signal-pro peptide sequence was replaced with murine beta-casein signal peptide sequence and the human t-PA variant mature peptide cDNA was inserted into the second exon of beta-casein gene. The fusion gene was microinjected in the fertilized mice eggs. A total of 285 embryos were microinjected and transferred into 13 surrogate mother mice. Twelve positive transgenic mice were identified through PCR and Southern blot analysis among 42 new born mice. Human t-PA variant was expressed in the milk of 7 transgenic mice, the highest expression level attained to 3.6593 micrograms/ml. The results demonstrated that the murine beta-casein gene regulatory elements can direct the human t-PA variant gene successfully express in the milk of transgenic mice. It lays great foundation for the research on the beta-casein knock-in mice mammary gland bioreactor model construction.

Expression of Smad4 Gene in Pichia pastoris and Identification of the Protein.

Smad4 is a novel tumor suppressor gene which is mutated or deleted in about 50% of pancreatic carcinoma and 30% of colon cancer. SMAD4 was expressed in Pichia pastoris and was characterized. The molecular weight of the expression product was shown to be about 67 kD, and 13 amino acids in N terminal were determined and were identical with the putative sequence from Smad4 cDNA, and it was able to specifically react with the antibody against SMAD4.