Creation And Validation Of An Isolator For The Rederivation Of Germ-free Genetically Engineered Mice

Genetically engineered animals are an important tool for understanding the function of host genes.However,with the advancement of multi-omics analysis techniques such as genomics and metabolomics,researchers have discovered that microorganisms have a significant impact on animals at the physiological,metabolic,and immunological levels.The microbiome has a complex connection with the host genome,and therefore,the presence of microorganisms cannot be ignored when studying functional genes.To better understand the interactions between microbes and host genes,germ-free genetically engineered mice have been used as a research model.However,the scarcity of these mice is due to the low efficiency of germ-free mice rederivation by hysterectomy and the difficulty of using embryo transfer techniques in polyvinyl chloride isolators.To address this issue,our study was conducted to create an isolator that could apply the embryo transfer technique to the production of germ-free mice.The main results of the study are as follows:1.In this study,an isolator was developed by combining a microscope with existing isolator technology.The researchers aimed to understand the function of each component of a conventional germ-free mice soft isolator by actively participating in the germ-free mice rederivation,breeding,and feeding in germ-free mice platform.This understanding served as the basis for designing the new isolator.The isolator operation module was divided into two sub-operating modules: module A,which consisted of the soft part,and module B,which consisted of the hard part.The operator utilized module A to breed and anesthetize sterile pseudopregnant mice,while module B was used for performing embryo transfer on the sterile pseudopregnant mice.To ensure the quality of the isolator,the researchers conducted field visits to laboratory animal cage apparatus companies to gain insights into cage apparatus production materials.Subsequently,they tested various materials for the isolator components and commissioned a foundry to produce the soft part of module A and the hard part of module B.Through physical assembly,some dimensional parameters were adjusted and corrected.Finally,this study involved the development and design of an isolator by understanding the function of each component of a conventional germ-free mice soft isolator,testing different materials,and making necessary adjustments to achieve the desired dimensions.2.This study developed a non-surgical technique for transferring embryos to germ-free mice.Pre-embryo manipulation was conducted in a customized embryo manipulation chamber located in an ultra-clean bench.Two-cell and blastocysts of C57BL/6 mice were obtained using in vitro fertilization with embryo in vitro culture techniques.The blastocysts were then aspirated through a transcervical embryo transfer device and packaged for transfer into the module A soft part.Subsequently,in the module B hard part,the pseudopregnant mice were anesthetized,and the embryos were transferred using the non-surgical embryo transfer technology.3.The results indicate that the isolator created in this study can establish a sterile environment.The method developed in this study for non-surgical embryo transfer in germ-free mice is reliable.This study employed Gram staining microscopy,brain-heart infusion broth culture method,liquid thioglycolate medium culture method,pancreatic casein soy broth liquid medium culture method,Columbia blood agar plate culture method,molecular biology techniques,parasite examination methods,virus examination methods,and sterile animal physiological characteristic analysis methods to assess various potential sources of contamination in the internal environment of isolators and during non-surgical embryo transfer of germ-free mice.In conclusion,this study has successfully developed an isolator that allows for the non-surgical embryo transfer of germ-free mice,creating a germ-free environment that is reliable and practical for use.This achievement is significant for improving the efficiency of rederiving genetically engineered germ-free mice and promoting research on the interactions between host genes and microorganisms.