Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestat...Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.展开更多
and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BL...and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BLG 5′ and 3′ flanking were the same as that of publication completely.The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2kb 5′ flanking with 2.1kb 3′ flanking.In order to assess the efficiency of BLG regulatory elements,green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47).Through observation under UV microscope and detection by fluorometer,it is demonstrated that the GFP has been successfully expressed in TD47 cell line.By virtue of direct observation and quantitative analysis,the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.展开更多
基金financially supported by the National Research Foundation of Korea(NRF-2021R1A5A1033157 for SRC program:382 Comparative medicine Disease Research Center,NRF-2021R1F1A105195313)the Research Institute of Veterinary Science,the BK21 Four for Future Veterinary Medicine Leading Education and Research Center,and a Seoul National University(SNU)grant(#550e2020005)。
文摘Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.
文摘and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BLG 5′ and 3′ flanking were the same as that of publication completely.The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2kb 5′ flanking with 2.1kb 3′ flanking.In order to assess the efficiency of BLG regulatory elements,green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47).Through observation under UV microscope and detection by fluorometer,it is demonstrated that the GFP has been successfully expressed in TD47 cell line.By virtue of direct observation and quantitative analysis,the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.
