A Preliminary Study On Transfection Of Fish Caudal Fin Cells And Mature Gametes By Electroporation

Electroporation transfection is a currently reliable method for preparing genetically modified organisms and studying the gene function of eukaryotic cells.It has the advantages of strong repeatability,convenient operation and suitable for many cell types.In principle,any cell can be obtained higher transfection efficiency by optimizing electrical parameters.However,relevant researches mostly focus on mammalian cells,and there are few reports on electrotransfection of fish cells in vitro.Fish conceive a large amount of eggs,but mature eggs do not have a good storage environment in vitro,so manipulation on fish eggs in vitro are often limited.Since the efficiency of electrotransfection is affected by many factors,such as electroporation parameters,plasmid concentration,cell type,etc.,how to optimize conditions of electrotransfection to obtain a better transfection effect is the most critical issue in the research and application of electroporation technology.In this study,we preliminary explored the conditions of electrotransfection and the temporary storage method of fish mature eggs in vitro by using crucian carp caudal fin cells and zebrafish mature gametes.The main results are as follows:1.The GFP and RFP fluorescent reporter plasmids are plasmid expression vectors commonly used to transfect mammalian cells.In order to verify whether the two fluorescent reporter plasmids can also be expressed in fish,the two plasmids were injected into one-cell zebrafish embryos by microinjection,and positive embryos with strong fluorescent expression were observed under a fluorescent microscope 24 hours later,indicating that both fluorescent reporter plasmids can be effectively expressed in fish.2.In order to establish the electrotransfection method of fish caudal fin cells,the GFP fluorescent reporter plasmid was used to transfect the cultured crucian carp caudal fin cells in vitro by electroporation,and the electrotransfection conditions of fish caudal fin cells were explored from voltage,pluse times,and plasmid concentration.The results showed that the voltage 60-70 v,pulses number 1-2,pulse length 55ms(capacitance 1100μF,resistance 50Ω),1mm electrode cup,plasmid concentration 500ng/μl,cell transfection efficiency can reach 70%.3.In order to extend the operation time of mature fish eggs,we explored the temporary storage method of mature fish eggs in vitro and independently developed an temporary storage solution for mature fish eggs in vitro,the formula is 68.25% basis culture medium + 1.75% carp serum + 30% fish ovary fluid,p H =8.1.The results showed that the embryo survival rate of mature zebrafish eggs stored in the temporary storage solution for 30 minutes and then in vitro fertilization was 90%.The morphology and structure of the egg envelope were observed by microscope and scanning electron microscopy(SEM).It is showed that the morphology and structure of zebrafish mature eggs were basically stable after 30 min in temporary storage solution.The thickness and pore size of the egg envelope were the same as those of the untreated eggs,while the egg envelope of the water-activated eggs became thinner and the pore size of the egg envelope increased significantly.4.RFP and GFP fluorescent reporter plasmids were respectively transfected into zebrafish sperm and mature eggs by electroporation,to explore the electrotransfection conditions of fish gametes.Zebrafish sperm were electrotransfected under the conditions of field strength 600～1000v/cm,pulse time 20 ms,pulse number 4,and every pluse interval 1s,one embryo similar to chimera embryo with red fluorescence was observed after in vitro fertilization.However,in the process of exploring the electrotransfection conditions of zebrafish mature eggs,no positive embryos with fluorescence expression have been observed.In summary,we preliminarily optimized electrotransfection conditions suitable for fish caudal fin cells cultured in vitro,which can make the cell transfection efficiency up to 70%;independently developed temporary storage solution for mature fish eggs in vitro;Meanwhile,we have preliminarily explored factors such as waveform,voltage,pulse number,and pulse time on the effect of fish gametes electrotransfection.Relevant research provides a technical approach to further improve the efficiency of fish cell transfection and the creation of genetically modified fish.