Recombinant adeno-associated virus （rAAV） vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequenc...Recombinant adeno-associated virus （rAAV） vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applica- bility to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer （SCNT）, this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subse- quently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.展开更多
Objective: TO investigate the killing differences of human blood group antibodies IgG and IgM on PBMC and RBC in peripheral blood of genetically modified pigs, and provide theoretical basis for the selection of pig bl...Objective: TO investigate the killing differences of human blood group antibodies IgG and IgM on PBMC and RBC in peripheral blood of genetically modified pigs, and provide theoretical basis for the selection of pig blood group for clinical application of porcine xenotransplantation. Methods: Serum samples were collected from 20 healthy subjects, 20 patients with end‑stage renal disease and 20 brain dead organ donors, and divided into 4 groups on the basis of ABO blood group (A: n=20;B: n=17;AB: n=7;O: n=16). Flow cytometry was used to detect antibody binding or complement dependent cytotoxicity test (CDC) between human serum and O blood group Wild‑type (WT), α1, 3‑galactosyltransferase gene‑knockout(GTKO), cytidine monophosphate‑N‑acetylneuraminic acid hydroxylase gene‑knockout (GTKO/β4GalNT2KO), β‑1, 4N‑acetylgalactosaminyltransferase gene‑knockout(GTKO/CMAHKO) and mononuclear cells (PBMC) and red blood cells (RBC) of Triple knockout (TKO/hCD55) porcine peripheral blood, respectively. Results: There was no significant difference in binding and killing of human serum antibodies with blood group A, B, AB and O on PBMC of WT pigs, GTKO pigs, GTKO/β4GalNT2KO pigs, GTKO/CMAHKO pigs and TKO/hCD55 pigs, respectively. There was no significant difference in RBC binding with RBC of WT pig, GTKO pig, GTKO/β4GalNT2KO pig, GTKO/CMAHKO pig and TKO/hCD55 pig. Conclusion: The selection of recipients of pigs with type O blood group can be done without considering blood group.展开更多
基金supported by the grants from the Danish National Researeh Infrastructure Programme to the Danish Genetieally Modified Animal Resource(DAG- MAR)as well as from the"Sino一Danish Breast Caneer Research Centre"under the ausPiees of the Danish National Researeh Foundation(Grundforskningsfonden)the National Natural Seience Foundation of China
文摘Recombinant adeno-associated virus （rAAV） vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applica- bility to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer （SCNT）, this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subse- quently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.
基金Key Science and Technology Project of Hainan Provincial Science and Technology Department (ZDKT2019009)Hainan Clinical Medical Center construction project.
文摘Objective: TO investigate the killing differences of human blood group antibodies IgG and IgM on PBMC and RBC in peripheral blood of genetically modified pigs, and provide theoretical basis for the selection of pig blood group for clinical application of porcine xenotransplantation. Methods: Serum samples were collected from 20 healthy subjects, 20 patients with end‑stage renal disease and 20 brain dead organ donors, and divided into 4 groups on the basis of ABO blood group (A: n=20;B: n=17;AB: n=7;O: n=16). Flow cytometry was used to detect antibody binding or complement dependent cytotoxicity test (CDC) between human serum and O blood group Wild‑type (WT), α1, 3‑galactosyltransferase gene‑knockout(GTKO), cytidine monophosphate‑N‑acetylneuraminic acid hydroxylase gene‑knockout (GTKO/β4GalNT2KO), β‑1, 4N‑acetylgalactosaminyltransferase gene‑knockout(GTKO/CMAHKO) and mononuclear cells (PBMC) and red blood cells (RBC) of Triple knockout (TKO/hCD55) porcine peripheral blood, respectively. Results: There was no significant difference in binding and killing of human serum antibodies with blood group A, B, AB and O on PBMC of WT pigs, GTKO pigs, GTKO/β4GalNT2KO pigs, GTKO/CMAHKO pigs and TKO/hCD55 pigs, respectively. There was no significant difference in RBC binding with RBC of WT pig, GTKO pig, GTKO/β4GalNT2KO pig, GTKO/CMAHKO pig and TKO/hCD55 pig. Conclusion: The selection of recipients of pigs with type O blood group can be done without considering blood group.
