Construction And Functional Identification Of Erythroid Specific Gypa-Cre Mouse Models

Purpose:Erythropoiesis is a process by which multipotential hematopoietic stem cells proliferate,differentiate and eventually form mature erythrocytes.Defective erythropoiesis leads to anemia which affects about one third of world's population.However,the mechanisms that regulate normal erythropoiesis or contribute to pathological erythropoiesis are only partially understood.Mouse models in which genes are conditionally manipulated in erythroid cells by the Cre/lox P recombination system provide powerful tools to the study of gene function in erythroid cells.It has been thought for a long time that erythropoietin receptor(Epor)is only expressed in erythroid cells and is a marker for erythroid lineage.Based on this assumption,a Epor-EGFPcre mouse model was generated and used to manipulate gene expression thought to be specifically in erythroid cells.However,studies from others and us have shown that Epor is expressed well beyond erythroid lineage.More recently,using a novel Epor-tdTomato-Cre mouse model we generated,we documented that Epor-Cre led to Cre-mediated recombination not only in erythroid cells but also in other cells such as erythroblastic island macrophages.Thus,erythroid specific Cre recombinase mouse model is urgently needed.Glycophorin A(Gypa)is a blood group antigen carrying protein that is abundantly and specifically expressed in erythroid cells.The goal of current study is to generate mouse models in which the expression of Cre recombinase and fluorescent protein EGFP or tdTomao is under the control of Gypa promoter.Such models will enable conditional gene manipulation in erythroid cells and facilitate studies on erythropoiesis.Methods:Firstly,NCBI and other databases were searched to query the location,full length,sequence and transcript,structural domain,regulatory region and other information of the Gypa gene,and targeting strategy of Gypa-EGFP-iCre and Gypa-tdTomato-iCre gene knock-in mouse models were designed;secondly,technical support was provided by the company and improved CRISPR/Cas9 gene editing technology was used to target and got positive F1 generation mice;then,the obtained Gypa-EGFP-iCre and Gypa-tdTomato-iCre mice were expanded and performed genotype identification,Western blot identification,fluorescence identification to confirm the expression of the foreign knock-in gene sequence,and body weight detection and blood routine parameter detection were carried out to confirm that the body weight and blood routine parameters were normal;further flow cytometry was used to detect the expression of Gypa in the erythroid and other blood cells;Gypa-EGFP-iCre^+/-mice were mated with Rosa26-loxp-stop-loxp-tdTomato^+/+mice and Gypa-tdTomato-iCre^+/-mice were mated with Rosa26-loxp-stop-loxp-EYFP^+/+mice,and then Rosa26-tdTomato^+/-Gypa-EGFP-iCre^+/-mice and Rosa26-EYFP^+/-Gypa-tdTomato-iCre^+/-mice were obtained,then flow cytometry was used to detect the fluorescence of tdTomato and EYFP in erythroid progenitors and late stage erythroid cells to detect the activity of Cre recombinase.Thus,erythroid specific Gypa-EGFP-iCre and Gypa-tdTomato-iCre mice that can be specifically used for erythroid related research were obtained.Results:1.Gypa-EGFP-iCre and Gypa-tdTomato-iCre gene knock-in mouse models were successfully constructed with Gypa as the target.Targeting strategies for Gypa-EGFP-iCre and Gypa-tdTomato-iCre gene knock-in mouse models were designed.Genotype identification,Western blot identification,and fluorescence identification of Gypa-EGFP-iCre and Gypa-tdTomato-iCre gene knock-in mice were performed to confirm the successful expression of the knock-in sequences.The body weight and blood parameters of the two mouse models had no significant difference between the heterozygous mice and WT mice.2.Gypa-EGFP and Gypa-tdTomato were specifically and highly expressed in the erythroid cells of Gypa-EGFP-iCre and Gypa-tdTomato-iCre knock-in mouse models.Flow cytometry results showed that in erythroid progenitors of Gypa-EGFP-iCre^+/-and Gypa-tdTomato-iCre^+/-mice,Gypa-EGFP and Gypa-tdTomato were partially expressed in BFU-E,and were expressed in CFU-E.And Gypa-EGFP and Gypa-tdTomato were highly expressed in the late stage erythroid cells and not expressed in other blood cells such as macrophages,megakaryocytes,monocytes,granulocytes,T cells and B cells.3.Cre recombinase in Gypa-EGFP-iCre and Gypa-tdTomato-iCre gene knock-in mouse models had high cutting efficiency.Flow cytometry results of expression of tdTomato and EYFP in etythroid progenitors and late stage erythroid cells in fetal liver and bone marrow of Rosa26-tdTomato^+/-Gypa-EGFP-iCre^+/-and Rosa26-EYFP^+/-Gypa-tdTomato-iCre^+/-mice showed that Cre recombinase could efficiently cut lox P sites in erythroid cells of Gypa-EGFP-iCre^+/-and Gypa-tdTomato-iCre^+/-mice.Conclusion:In conclusion,Gypa-EGFP-iCre and Gypa-tdTomato-iCre gene knock-in mouse models were successfully constructed.Gypa-EGFP and Gypa-tdTomato were specifically and highly expressed in erythroid cells but were not expressed in a variety of non-erythroid blood cells.Cre recombinase had high efficiency in erythroid cells.Therefore,erythroid specific Gypa-EGFP-iCre and Gypa-tdTomato-iCre mouse models we constructed will provide erythroid specific conditional gene editing tools for studies on erythropoiesis,and also provide erythroid specific mouse models for researchs based on erythroid cell tracing.