Study On The Effect Of Carboxymethyl Cellulose On Transfection Effect Of Viral Vectors In Chicken Embryos

Transgenic technology has developed so far,people continue to develop new methods to prepare transgenic chickens,viral vector method is currently the most commonly used method for preparing transgenic chickens,but the method has relatively low efficiency defects.In order to improve the transfection efficiency of chicken embryos by viral vector,an enhancing reagent for the preparation of transgenic chickens by viral vector method was developed,and the following tests were carried out in this study:1.By comparing the transfection efficiency of lentiviral vectors carrying enhanc green fluorescent protein(EGFP)with liposome-mediated EGFP plasmid vectors on chicken embryo fibroblasts(CEF)cultured in vitro,it was found that viral vectors were more efficient than liposome-mediated plasmid vectors for CEF cells Microinjection of viral vector buffers containing 0.036%,0.1%,and0.36% carboxymethylcellulose(CMC,a thickener in the food industry)in the subleopmental cavity of chicken embryos to the X stage was found to significantly increase the residence time of the buffer in the subleople of the embryo,and the results suggested that CMC may improve its transfection effect by prolonging the time of action of the viral vector on chicken embryos.2.In order to explore whether CMC affects the transfection efficacy of the viral vector itself,0.3 μL,1 μL,3 μL,3 μLEGFP lentivirus or EGFP adenovirus were added to the HEK293 T cell culture medium containing 0.36%,0.1% and 0.036% CMC,respectively,and it was found that 0.036% CMC would not affect the transfection efficiency of EGFP lentivirus to HEK293 T cells.All of the remaining concentrations of CMCs significantly reduced the transfection efficiency of EGFP lentivirus and EGFP adenovirus to HEK293 T cells,and the decrease increased with CMC concentrations,and 0.3 μL,1 μL,3 μL,3μLGFP lentivirus or EGFP adenovirus were added to the CEF cell culture medium containing concentrations of 0.36%,0.1%,and 0.036% CMC,respectively,and found that in addition to individual experimental groups,All of the above concentrations of CMC significantly reduced the transfection efficiency of CEF cells by EGFP lentivirus and EGFP adenovirus to varying degrees.3.The EGFP lentiviral solution and EGFP adenovirus liquid containing 0.1% CMC were microinjected into the X stage chicken embryos,and it was found that the survival rate of the chicken embryos was not significantly affected after the addition of 0.1% CMC.Microinjection of EGFP lentiviral fluid and EGFP adenovirus fluid containing 0.036%,0.1%,and 0.36% CMC into stage X chicken embryos was found to increase the proportion of fluorescent chicken embryos at all concentrations,but the difference was not significant;however,after counting the number of chicken embryos with the highest fluorescence expression in each group,it was found that the addition of 0.1% CMC to the EGFP adenovirus injection group could significantly increase the number of chicken embryos with the highest fluorescence expression.The above results show that although CMC will reduce the transfection efficiency of the virus vector to cells to a certain extent,CMC can prolong its action time in chicken embryos and ultimately improve its transfection effect on chicken embryos.This study further improves the viral vector method and lays the foundation for better preparation of transgenic chickens.