Establishment Of Tdtomato Transgenic Mouse Lineages And Their Application In Cell Tracing

Objective:1.To establish transgenic mouse lineages that can stably express the tandem-dimer tomato(tdTomato)gene and to observe the level of fluorescence expression in tissue sections.Detect physiological and biochemical indicators of tdTomato transgenic mice to ensure that they meet the standards of experimental mice;2.The fluorescence expression of neural stem cells from tdTomato-positive mice were observed in the tracer experiments.An initial attempt was made to co-culture experiments of two-color allogeneic stem cells in order to explore the stability of fluorescence expression during cell culture and differentiation.Methods:The experiment is divided into two parts.In the first part,the gateway clone technique and DNA microinjection were used to construct fertilized ova containing the tdTomato expression vector,which were then transferred to pseudopregnant mice for natural delivery.tdTomato-positive mice were identified and picked using a two-way verification method for breeding and establishment.The biochemical indexes and growth characteristics of the tdTomato transgenic mice were compared with the wild-type mice.In the second part,the neural stem cells of tdTomato transgenic mice were extracted and cultured to three generations,and then transplanted to the spinal cord injury site of the model mouse for tracer observation.In addition,bone marrow mesenchymal stem cells and neural stem cells from eGFP transgenic mice were co-cultured with neural stem cells from tdTomato transgenic mice.The differentiation of stem cells and the expression of two kinds of fluorescence were observed.Results:The tdTomato transgenic mice were successfully constructed,and their lines were established.The biochemical indexes and growth characteristics of the tdTomato transgenic mice did not significantly differ from those of the wild-type mice.Red fluorescence was observed in the tissue sections and brain-derived neural stem cells under fluorescence microscopy.Significant fluorescence was observed at the injection site in the tracer experiments,and two-color fluorescence was stably expressed in the co-culture experiments.Conclusion:Stable red fluorescence occurred in the tissues and cells of the tdTomato transgenic mice,and the positive rate will increase with generations.Red fluorescence of tdTomato transgenic mice was not affected by the routine experimental operations,and tdTomato transgenic mice can support the experimental requirements of cell culture in vitro and two-color co-culture.The above experimental results show that tdTomato transgenic mice are a more convenient and practical fluorescent tool mouse,which can be applied to research the direction of differentiation after stem cell co-culturing.