[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cells (EPCs) seeded on biomaterials can effectively promote diabetic ischemic wound healing. However, the function of transplanted EPCs is negatively affected by a high-glucose and ischemic microenvironment. Our experiments showed that EPC autophagy was inhibited and mitochondrial membrane potential (MMP) was increased in diabetic patients, while adenosine treatment decreased the energy requirements and increased the autophagy levels of EPCs. In animal experiments, we transplanted a biomaterial seeded with EPCs onto the surface of diabetic wounds and found that adenosine-stimulated EPCs effectively promoted wound healing. Increased microvascular genesis and survival of the transplanted cells were also observed in the adenosine-stimulated groups. Interestingly, our study showed that adenosine increased the autophagy of the transplanted EPCs seeded onto the biomaterial and maintained EPC survival at 48 and 96 hours. Moreover, we observed that adenosine induced EPC differentiation through increasing the level of autophagy. In conclusion, our study indicated that adenosine-stimulated EPCs seeded onto a biomaterial significantly improved wound healing in diabetic mice; mechanistically, adenosine might maintain EPC survival and differentiation by increasing high glucose-inhibited EPC autophagy and maintaining cellular energy metabolism.
[Show abstract][Hide abstract] ABSTRACT: The application of tissue-engineered blood vessels (TEBVs) is the main developmental direction of vascular replacement therapy. Due to few and/or dysfunctional endothelial progenitor cells (EPCs), it is difficult to successfully construct EPC capture TEBVs in diabetes. RNA has a potential application in cell protection and diabetes treatment, but poor specificity and low efficiency of RNA transfection in vivo limited the application of RNA. Based on acellular vascular matrix, we proposed an aptamer-siRNA chimeras modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consisted of two parts, CD133-adenosine kinase (ADK) chimeras and TEBV scaffold. Our results showed that CD133-ADK chimeras could selectively capture the CD133-positive cells in vivo, and then captured cells internalized the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK siRNA by dicer, resulting in depletion of ADK. ADK deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce non-specific RNA transfection, we increased the proportion of HAuCl4 during the material preparation, through which the transfection capacity of polyethyleneimine (PEI)/polyethylene glycol (PEG)-capped gold nanoparticles (PEI/PEG-AuNPs) was significantly decreased, and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2' O-Methyl (2'-OMe) modification was used to enhance the in vivo stability of RNA chimeras. At day 30 post-grafting, the patency rate of CD133-ADK chimeras-modified TEBVs reached 90% in diabetic rats and good endothelialization was observed.
[Show abstract][Hide abstract] ABSTRACT: Small-diameter tissue-engineered blood vessels (TEBVs) have been associated with low long-term patency rates primarily because of acute thrombosis in early stages and an inability to achieve early endothelialization. Platelets and endothelial progenitor cells (EPCs) play a key role in these processes. A nano delayed-release aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR)-bound tissue-engineered blood vessel was implanted in rat carotid arteries for three months. AICAR-bound TEBVs had high patency rate compared with control TEBVs after 3 mouths. We found that AICAR maintained moderate platelet aggregation in vivo. In vitro data indicated that AICAR inhibits the release of 5-hydroxytryptamine and thromboxaneA2 in activating platelets to reduce platelet aggregation. Then, we confirmed that AICAR strengthens the EPCs energy state, which results in earlier endothelialization. The homing, migration, and paracrine function of EPCs were enhanced by AICAR in vitro. Besides, AICAR can contribute to the migration of endothelial cells near the anastomosis. The cellularization of TEBVs at different time points was observed too. In conclusion, our study suggests the application of nano delivery material containing AICAR can effectively improve small-diameter tissue-engineered blood vessels by maintaining moderate platelet aggregation and improving metabolism of EPCs.
Tissue Engineering Part A 03/2015; 21(13-14). DOI:10.1089/ten.TEA.2015.0013 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A tissue-engineered blood vessel (TEBV) modified with CS/β-CD nanoparticles is constructed, effectively controling the release of adenosine, and this type of TEBV can keep open for over 6 months. This study also demonstrates for the first time that adenosine promotes endothelial progenitor cell (EPC) mobilization and homing via energy conversion, achieving rapid endothelialization of TEBV.
[Show abstract][Hide abstract] ABSTRACT: Great challenges in transplantation of mesenchymal stem cells (MSCs) for treating ischemic diabetic ulcers (IDUs) are to find a suitable carrier and create beneficial microenvironment. Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, is considered angiogenic and neuroprotective. Given that IDUs are caused by vascular disease and peripheral neuropathy, we used BDNF as a stimulant, and intended to explore the role of new biomaterials complex with MSCs in wound healing. BDNF promoted the proliferation and migration of MSCs using MTT, transwell and cell scratch assays. The activity of human umbilical vein endothelial cells (HUVECs) was also enhanced by the MSC-conditioned medium in the presence of BDNF, via a VEGF-independent pathway. Because proliferated HUVECs in the BDNF group made the microenvironment more conducive to endothelial differentiation of MSCs, by establishing co-culture systems with the two cell types, endothelial cells derived from MSCs increased significantly. A new biomaterial made of polylactic acid, silk and collagen was used as the carrier dressing. After transplantation of the BDNF-stimulated MSC/biomaterial complex, the ulcers in hindlimb ischemic mice healed prominently. More blood vessel formation was observed in the wound tissue, and more MSCs were co-stained with some endothelial-specific markers such as CD31 and von Willebrand Factor (vWF) in the treatment group than in the control group. These results demonstrated that BDNF could improve microenvironment in the new biomaterial, and induce MSCs to differentiate into endothelial cells indirectly, thus accelerating ischemic ulcer healing.
Tissue Engineering Part A 10/2014; 21(5-6). DOI:10.1089/ten.TEA.2014.0113 · 4.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This investigation was aimed to explore whether over-expression of 27heme oxygenase-1 (HO-1) could protect bone marrow mesenchymal stem cells(BMSCs)against injury induced by high-concentration glucose. We cultured BMSCs in high-concentration glucose medium, and up-regulated or inhibited HO-1 expression in BMSCs through its agonist or inhibitor. We detected the ability of BMSCs proliferation and secretion respectively by MTT and enzyme-linked immunosorbnent assay (ELISA). Then we detected the effect of BMSCs conditions medium on proliferation and migration of human umbilical vein endothelial cells (HUVECs) through scratch experiments and transwell assay. It was found that HO-1 over-expression could not only promote BMSCs proliferation, but also promote secretion of vascular endothelial growth factor (VEGF), and could further accelerate the proliferation and migration of HUVECs. It could be well concluded that HO-1-over-expressing BMSCs can not only inhibit damage induced by high-concentration glucose, but can promote the proliferation and migration of vascular endothelial cells through paracrine as well. The result indicated that HO-1-over-expressing BMSCs played an important role in the treatment of diabetic vascular complication.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 08/2013; 30(4):798-802.
[Show abstract][Hide abstract] ABSTRACT: Diabetic ischemic ulcer is an intractable diabetic complication. Bone marrow mesenchymal stem cells (BMSCs) have great potential in variety of tissue repair. In fact, poor cell viability and tolerance limit their ability for tissue repair. In addition, it is difficult for stem cells to home and locate to the lesion. In this study, we explore whether over-expression of heme oxygenase-1 (HO-1) in BMSCs complexed with collagen play an important role in treatment of diabetic ischemic ulcers. In vitro, over-expression of HO-1 promoted the proliferation and paracrine activity of BMSCs and the conditioned medium of BMSCs accelerated HUVECs migration and proliferation. These processes were closely related to Akt signaling pathway and were not dependent on Erk signaling pathway. In vivo, in order to make BMSCs directly act on the wound, we choose a solid collagen as a carrier, BMSCs were planted into it, ischemic wounds of diabetic mice were covered with the complex of BMSCs and collagen. The results indicate that the complex of HO-1-overexpressing BMSCs and collagen biomaterials can significantly promote angiogenesis and wound healing. These preclinical findings open new perspectives for the treatment of diabetic foot ulcers.
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1(-/-)) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1(-/-) mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1(-/-) mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1(-/-) mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1(-/-) mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.
[Show abstract][Hide abstract] ABSTRACT: The patency rate of small-diameter tissue-engineered blood vessels is the determinant for their application in coronary artery bypass grafting. The coronary artery is innervated by vagus nerves. The origin of vagus nerves is rich in brain-derived neurotrophic factors (BDNF). We have investigated whether BDNF could improve the patency rate of small-diameter tissue-engineered blood vessels through promoting stem cell homing and paracrine activity. In vitro, we isolated early and late endothelial progenitor cells (EPCs) and found BDNF could promote single clone formation and paracrine function of EPCs, and could also induce the proliferation, migration and differentiation of late EPCs. BDNF could enhance the capturing of EPCs in parallel-plate flow chamber. Flow cytometric analysis and laser-scanning confocal microscope showed BDNF could enhance the mobilization and homing of C57BL/6 mouse EPCs after wire injury. Based on it, BDNF was coupled to the lumen surface of the blood vessel matrix material incubated with collagen through SPDP to construct BDNF-modified small-diameter tissue-engineered blood vessel. The blood vessel patency rate was significantly higher than that of control group 8 weeks after implantation in rats and the endothelialization level was superior to control. These results demonstrate that BDNF can effectively improve patency of small-diameter tissue-engineered blood vessels through stem cell homing and paracrine, and it is expected to play an important role in the construction of substitutes for coronary artery bypass grafting.
[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cells (EPCs) mobilization and homing are critical to the development of an anti-thrombosis and anti-stenosis tissue-engineered blood vessel. The growth and activation of blood vessels are supported by nerves. We investigated whether nerve growth factors (NGF) can promote EPCs mobilization and endothelialization of tissue-engineered blood vessels. In vitro, NGF promoted EPCs to form more colonies, stimulated human EPCs to differentiate into endothelial cells, and significantly enhanced EPCs migration. Flow cytometric analysis revealed that NGF treatment increased the number of EPCs in the peripheral circulation of C57BL/6 mice. Furthermore, the treatment of human EPCs with NGF facilitated their homing into wire-injured carotid arteries after injection into mice. Decellularized rat blood vessel matrix was incubated with EDC cross-linked collagen and bound to NGF protein using the bifunctional coupling agent N-succinmidyl3-(2-pyridyldit-hio) propionate (SPDP). The NGF-bound tissue-engineered blood vessel was implanted into rat carotid artery for 1 week and 1 month. NGF-bound blood vessels possessed significantly higher levels of endothelialization and patency than controls did. These results demonstrated that NGF can markedly increase EPCs mobilization and homing to vascular grafts. Neurotrophic factors such as NGF have a therapeutic potential for the construction of tissue-engineered blood vessels in vivo.
[Show abstract][Hide abstract] ABSTRACT: To use the mice deficient in both adenosine receptor A(2A)(A(2A)R(-/-)) and apolipoprotein E (apoE(-/-)) to investigate the role of A(2A)R in mediating the interactions of leukocytes with injured arterial walls and the formation of arterial neointima induced by a guide wire.
In apoE(-/-) mice, A(2A)R deficiency increased the size of the arterial neointima in injured carotid arteries by 83%. Arterial neointima formation was also enhanced in chimeric mice that underwent bone marrow transplantation (these mice lacked A(2A)R in their bone marrow-derived cells). Epifluorescence intravital microscopy showed that neutrophil rolling and adherence to the injured arterial area were enhanced by 80% and 110% in A(2A)R(-/-)/apoE(-/-) mice, respectively. This phenomenon occurred even though the protein levels of homing molecules on A(2A)R-deficient neutrophils were unchanged from those of wild-type neutrophils. A(2A)R-deficient neutrophils exhibited an increase in the phosphorylation of p38 mitogen-activated protein kinase, P-selectin glycoprotein ligand-1 (PSGL-1) clustering, and the affinity of b(2) integrins. The inhibition of p38 phosphorylation abrogated the increased PSGL-1 clustering and beta(2) integrin affinity, thus reversing the increased homing ability of A(2A)R-deficient leukocytes.
A(2A)R plays a complex role in inflammation and tissue injury. The deficiency of A(2A)R enhances the homing ability of leukocytes and increases the formation of the arterial neointima after injury. A(2A)R antagonists are being tested for the treatment of neurodegenerative and other chronic diseases. An evaluation of the effect of A(2A)R antagonists on arterial restenosis after arterial angioplasty should be conducted.
[Show abstract][Hide abstract] ABSTRACT: Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation.
Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation.
Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.
[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cell (EPC)-seeded intravascular stents may reduce or prevent in-stent restenosis. A20 can play an important role for preventing vascular restenosis. Therefore, it is very important how to enhance the seeding efficiency of A20-modified EPCs on the stent for preventing in-stent restenosis. To approach this problem, we developed a novel transgenic EPC-seeded stent and evaluated its feasibility and efficiency. EPCs were isolated and purified from umbilical blood using immunomagnetic beads and then transfected with the A20 gene. One stent type (type 1) was coated with EDC cross-linked collagen, and another stent type (type 2) was coated with EDC cross-linked collagen and bound to the CD34 antibody using the bifunctional coupling agent N-succinmidyl3-(2-pyridyldithio) propionate (SPDP). Then, the stents were seeded with EPCs transfected with the A20 gene. The stents were implanted in biological artificial vessels, and cell adhesion was determined in a flow chamber. Cell growth was also measured. EPCs were transfected successfully with the A20 gene. The cells covered both types of stents with favorable biological function. After placement in a flow chamber, the number of cells attached to type 1 stents significantly dropped and their distribution was scattered. Type 2 stents were basically covered with cells and there were more cells on type 2 stents than on type 1 stents (p < 0.01). Collagen-coupled antibody effectively improves the seeding of transgenic EPCs, offering a new choice of stents to prevent restenosis caused by vascular disease after interventional treatment.
Journal of Biomedical Materials Research Part A 11/2009; 91(2):623-8. DOI:10.1002/jbm.a.32300 · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall. The A(2A) receptor (A(2A)R) plays a central role in many antiinflammatory effects of adenosine. However, the role of A(2A)R in atherosclerosis is not clear.
The knockout of A(2A)R in apolipoprotein E-deficient (Apoe(-/-)/A(2A)R(-/-)) mice led to an increase in body weight and levels of blood cholesterol and proinflammatory cytokines, as well as the inflammation status of atherosclerotic lesions. Unexpectedly, Apoe(-/-)/A(2A)R(-/-) mice developed smaller lesions, as did chimeric Apoe(-/-) mice lacking A(2A)R in bone marrow-derived cells (BMDCs). The lesions of those mice exhibited a low density of foam cells and the homing ability of A(2A)R-deficient monocytes did not change. Increased foam cell apoptosis was detected in atherosclerotic lesions of Apoe(-/-)/A(2A)R(-/-) mice. In the absence of A(2A)R, macrophages incubated with oxidized LDL or in vivo-formed foam cells also exhibited increased apoptosis. A(2A)R deficiency in foam cells resulted in an increase in p38 mitogen-activated protein kinase (MAPK) activity. Inhibition of p38 phosphorylation abrogated the increased apoptosis of A(2A)R-deficient foam cells.
Inactivation of A(2A)R, especially in BMDCs, inhibits the formation of atherosclerotic leisons, suggesting that A(2A)R inactivation may be useful for the treatment of atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: Core2 1 to 6-N-glucosaminyltransferase-I (C2GlcNAcT-I) plays an important role in optimizing the binding functions of several selectin ligands, including P-selectin glycoprotein ligand. We used apolipoprotein E (ApoE)-deficient atherosclerotic mice to investigate the role of C2GlcNAcT-I in platelet and leukocyte interactions with injured arterial walls, in endothelial regeneration at injured sites, and in the formation of arterial neointima.
Arterial neointima induced by wire injury was smaller in C2GlcNAcT-I-deficient apoE(-/-) mice than in control apoE(-/-) mice (a 79% reduction in size). Compared to controls, apoE(-/-) mice deficient in C2GlcNAcT-I also demonstrated less leukocyte adhesion on activated platelets in microflow chambers (a 75% reduction), and accumulation of leukocytes at injured areas of mouse carotid arteries was eliminated. Additionally, endothelial regeneration in injured lumenal areas was substantially faster in C2GlcNAcT-I-deficient apoE(-/-) mice than in control apoE(-/-) mice. Endothelial regeneration was associated with reduced accumulation of platelet factor 4 (PF4) at injured sites. PF4 deficiency accelerated endothelial regeneration and protected mice from neointima formation after arterial injury.
C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation, and this effect is attributable to decreased leukocyte recruitment to injured vascular walls and increased endothelial regeneration. Both C2GlcNAcT-I and PF4 are promising targets for the treatment of arterial restenosis.
[Show abstract][Hide abstract] ABSTRACT: To determine if A20, a zinc finger protein that mediates the inflammatory response, affects monocyte-endothelial cell-cell interactions induced by low shear flow.
Primary cultured endothelial cells (EC) were transfected with an A20 expression vector, and the VCAM-1, ICAM-1 and IL-8 mRNA, and protein expression levels in A20-transfected EC lysates were checked by PCR array and ELISA, respectively. CD14-positive monocyte migration toward and adhesion to EC were measured using a parallel plate flow chamber.
Low shear stress, defined as 0.2 Pa for 6 h, normally increases VCAM-1, ICAM-1 and IL-8 expression in EC and allows for binding of monocytes to EC. We found that overexpression of A20 in EC inhibits their normal expression of VCAM-1, ICAM-1 and IL-8 under low shear stress conditions. We also found that A20 inhibits IkappaBalpha degradation in EC following low shear stress exposure and that it attenuates the rolling and EC adhesive properties of shear-induced monocytes as compared with untransfected control EC. The results demonstrate that A20 overexpression in EC inhibits low shear flow-induced monocyte-EC interactions. These findings may be useful in the development of therapeutic agents aimed at treating chronic inflammatory diseases like atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether decellularized vascular tissues and A20-regulated endothelial progenitor cells can be used for constructing a transgenic tissue-engineered blood vessel with anti-atherosclerotic vascular stenotic properties. A20 gene-transfected endothelial progenitor cells differentiated endothelial cells and smooth muscle cells attached to and migrated into the decellularized porcine vascular scaffolding in a bioreactor. The histology of the conduits revealed viable and layered tissue. Scanning electron microscopy showed confluent, homogeneous tissue surfaces. The mechanical strength of the pulsed constructs was similar to that of the human artery. In vivo, the A20 gene-transfected tissue-engineered blood vessels were transplanted into the carotid artery of a rat for 6 months. Blood vessel xenotransplantation caused hyperacute rejection; all transplanted control blood vessels were completely rejected, but A20-transfected tissue-engineered blood vessels demonstrated good flow on implantation, and remained open for 6 months postoperatively, as assessed by Doppler. The HE stain demonstrated that the vessels were patent, without evidence of stenosis or dilatation after 6 months. These results demonstrate that transgenic tissue-engineered blood vessels have long-term patency and unique anti-stenotic properties.
[Show abstract][Hide abstract] ABSTRACT: This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 07/2008; 25(3):662-7.
[Show abstract][Hide abstract] ABSTRACT: Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.
In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.
It is feasible to design specific zinc finger proteins by bioinformatics methods.