A Novel Noninvasive Delivery Of DNA Into Zygotes By AuNPs-PEI For Transgenic Mouse

The technique by which known foreign genes are integrated into the genomes of eukaryotic cells and expressed stably is called gene introduction,which is the fundamental way to change the genetic traits of organisms.At present,microinjection has become a very widely used basic technique because of its relatively stable effect and high success rate of gene introduction.However,achieving these advantages requires skilled technicians and expensive laboratory equipment.At the same time,oocytes are prevented from being damaged and kept safe by delicate manipulation and precise control of the amount of DNA injected,but only be injected one by one,with a limited number of samples to process.Therefore,the traditional method of introducing exogenous genes into embryonic cells can no longer meet the current research needs,especially the research progress of CRISPR/Cas9 gene editing and RNA interference gene silencing urgently needs a new method to replace the traditional method.A transgenic system of noninvasive cytosolic trans-port based on exogenous plasmid DNA(pIRES2@EGFP)loading on gold nanoparticles(AuNPs)coated with polyethyleneimine(PEI)was successfully constructed,which can remove the technical threshold and possible damage of microinjection to oocytes and embryo development.In this study,mouse zygotes were cultured in KSOM medium containing the complex of AuNPs@PEI@DNA(APD)until formation of blastocyts,and then transferred into pseudopreg-nant female mice for obtaining transgenic offspring.Fluorescence observations confirm the expression of GFP in early embryos;importantly,data from identifying experiments by genotyping,immunofluorescence and DNA sequencing show that the GFP sequence indeed integrates into the chromosome of offspring individuals and is expressed.Furthermore,TUNEL test and analysis on cell differentiation of blastocysts indicate an excellent safety of APD to the develop-ment of embryos.All these results suggested that AuNPs,being an alternative technique to traditional microinjection,can efficiently introduce biomolecules into early embryonic cells without the prerequisite of expensive micromanipulators and skilled technicians,which will greatly promote study of life sciences in various aspects.