Construction Of Vector Tandem Expressing Shrnas Specific To Cardiac Kir2.1 Gene And Its Effect In Vitro

Backgand Currently electronic pacemakers are the main therapy for heart block and other electrophysiological abnormalities.But it has a lot of shoutcomings:limited battery life,the need for permanent catheter implantation into the heart,and lack of reponse to autonomic neurohumours.Furthermore,they are not suitable for patients with high risk of infection or too young.Therefore the traditional electronic pacemaker is not the optimal.In terms of both physiological function of the heart and adaptability to the human body the best pacemaking method would be the biological pacemaker.The search for a biological pacemaker centered on three gene therapy strategies:(1)upregulate the β₂-adrenergic receptors by transfecting cloned receptor gene that increase heart rate; (2)transfer α or β subunits of the human pacemaker current to induce autonomical responsive pacemaker function in ventricular myocytes ; (3)change the balance of potassium current in ventricular myocytes and make them get pacemaking function. Upregulation of the β₂ -adrenergic receptors by gene transfection is modulate the native pacemaker cells rather than the pacemaker itself.The transfection of the genes expressing α or β subunits of the human pacemaker current to induceautonomically responsive pacemaker function in ventricular myocytes can be done in two ways:one is transfect the myocytes by viral vectors;one is transplant mesenchymal stem cells or embryonic stem cells which were tranfected by a or psubunits of the human pacemaker current.Both methods can get only low beating rate biological pacemaker electrocardiography by vagal stimulation to suppress the sinus rhythm.Therefore these methods are not suitable for making of biological pacemaker.In 2002 ,Miake reported a biological pacemaker created by dominant-negative , they got spontaneous ventricular rhythm which was more rapid than the physiologyical sinus pacemaker. This provided proof of concept for biological pacemaking.Silva J and Rudy Y studed the mechanism of the biological pacemaker,and found that after 81% Ikl suppression,the cardiac myocytes will generate a spontaneous action potential, the Na-Ca2 exchanger is the pacemaking current in these cardiac myocytes,the beating rate of these cardiac myocytes increased with the degree of suppression of the Ikl,and they are responsive to the (3-adrenergic stimulation.Therefore suppression Ikl of the cardiac myocytes Ikl is the best method to creat biological pacemaker now.So,we design a study of suppression of kir2.1 gene by RNA interference(RNAi) technology.RNAi is a powerful molecularbiologial technology for knock-down genes in recent years.The suppression of RNAi to the target genes is highly specific and stable.The RNAi has an amplification effect,and can suppress the target genes very quickly.The effect can be observed 48 hours after the cells were transfected,and 10 genes can be knocked-down 10 genes in one week.There are two RNAs being used in the RNAi: siRNA(small interference RNAs) and shRNA(short hairpin RNAs).The shRNA is more effective than the siRNA.The RNAi has site-effect,there is no good principle for chosing the best targets.People usually chose five targets and then screen the best one by exprement.This is very difficult.Therefore we design a vector tandem expressing five shRNAs.The reconstruct vector can expression five shRNAstargeting five sites of rat kir2.1 gene.This can save the screen time and get the best effect of suppression of the gene.In this study,we constructed a vector tandem expressing five shRNAs of the rat kir2.1,and then transfected the vector into the cultured rat cardiac myocytes.The expression of the rat kir2.1 gene was detected by RT-PCR and Western-blot,and the change of the beating rate of the cardiac myocytes observed.This will give evidence for creating a biological pacemaker by RNAi in rat in vivo,and will give a new strategy for the development of biological pacemakers.There is no report about creating a boilogial pacemaker by RNAi untill now.Part 1 Construction of vector tandem expressing shRNAs specific to kir2.1 gene of rat cardiomyocytesObjective To construct an eukaryotic vector tandem expressing five shRNAs specific to rat cardiomyocytes kir2.1 gene.Methods To select five RNAi sites targeting the rat kir2.1 gene,designed and synthesized five pairs of oligonuleotides fragments Kl, K2> K3n K4 and K5,annealed them to double-strand ,then cloned them into the vectors pGenesil-1 > pEGFP6-l -. pG6-3 > pG6-4 which containing U6 promoters.Excised two of these recombinant vectors then cloned Kl and K2 into one vector, clonded K3 and K4 into another one.Then excised these two vectors and cloned the four genes into one vector named pEGFP6- 1K1 +K2 +K3 +K4.Finally excised pEGFP6-l K1+K2+K3+K4 and another vector containg K5 and cloned five genes to one vector,named pEGFP6-lkir2.1 .The recombinant vectors were identified by restriction enzyme digestion and assayed sequence of them.Results Restrictive enzyme digestion analysis showed that every recombinant eukaryotic expression vectors containing the aim genes had been constructed successfully. Sequencing result revealed the sequence of the vectors were all right.Part 2 Study on the transfection efficiency of eukaryotic vector tandem expressing shRNAs specific to rat kir2.1 gene mediated by cationic liposomeObjective To investigate the efficiency of of eukaryotic vector tandem expressing five shRNAs specific to rat kir2.1 by cationic liposome,and find a best ratio of liposome to vector for transfecting rat cardiomyocytes.Methods (l)The rat ventricular muscles digested and myocytes isolated and cultured.The myocytes were identified by immunohistochemical stain ,meanwile detected the purity of the myocytes.(2)Accodding to the volume of the liposome and the weight of the vector,four ratioes of liposome/vector transfection liquid were prepared: 1:1,2:1,3:1 and 4:1.After transfected the myocytes,the toxicity of the transfection liquid were tested by trypan blue stain.The expression of EGFP was observed under fluorescence microscope and the transfection efficiency was detected by flow cytometry also.(3)Cardiac myocytes viability and death rate was determined with trypan blue.Results (l)The lively ratio of mycyotes was 86.5% and its purity ratio was 95%.(2)There were a few of myocytes expressed EGFP 12h later,and more myocytes expressed EGFP after 48h and 72h.(3)The death rate of the myocytes was the highest when the ratio of liposome/vector was 4:1,which was markedly higher than that in the other three groups (p<0.01) .(4)The transfection efficiency was the highest when the liposome/vector ratio was 3:1, which was markedly higher than that in the other three groups( p<0.01).Part 3 The inhibition of kir2.1 expression in cultured rat ventricular myocytes by RNAi and its effect in vitroObjective To investigate the inhibition of kir2.1 expression in cultured rat ventricular myocytes by RNAi and observe the effect on beating rate of them.Methods The Wistar rat ventricular myocytes were isolated,cultured and divided into three groups:test group(T),negative plasmid group(Cl) and normal contonl group(CO).then transfected these myocytes with the plasmid pEGFP6-lkir2.1 which expressing five shRNAs specific to rat kir2.1 gene.The transfection was mediated by liposome,the ratio of liposome/plasmid was 3:1.The beating rate was counted under inverted microscope after transfection.RT-PCR and Westrrn-blot were carried out to detect the expression of the mRNA and protein of kir2.1.Results (1) After 24h of transfection,the myocutes beating rate of group T and Cl was lower than that in group CO (p<0.01) ,there was no diffenence between group T and group Cl. After 48h of transfection,the myocytes beating rate in group T and group Cl become faster (p<0.01) ,which in group T was much more faster than that in group Cl (p<0.01 ) ,but there were no difference between group Cl and group CO . After 72h of transfection,the beating rate in group T was much faster than 48h( p<0.01),while the beating rate in group Cl and CO was remain unchanged. After 96h of transfection, the beating rate of every group was same to the 72hs.(2)The expression of mRNA of kir2.1 of group T was markedly lower than that of group Cl and CO (p<0.01) .(3) The expression of the kir2.1 protein of group T was markedly lower than that of group Cl and CO (p<0.01) .Conclusions1. The construction of plasmid expressing five shRNAs specific to rat kir2.1 gene established the basis for studying biological pacemaking by RNAi.2. Isolating neborn rat cardiaomyosytes used trypsin could get a high viability .Appled differential adhesion method and added Brdu into the MEDM in the first few days could increase the purity of the cardiomyocytes.3. Liposome is a good vector for mediating plasmid transfect into ventricular myocytes.The best ratio of Metefectane to pEGFP6-lkir2.1 for transfecting ventricular myocytes was 3:1.4. RNAi can suppress the expression of the mRNA and protein of kir2.1.5. RNAi can enhance the rhythmic ability of ventricular myocytes and increase the beating rate of them.Therefore the RNAi technique maybe a mew method for biological pacemaking.