Ng ZFN (Z891) were cotransfected into HEK293 cells, hygromycin selection was performed by culturing the cells in the presence of 2 mg/ml hygromycin B for two days. The selected or unselected (control) cells were plated at a density of 3,000 cells/100 mm dish,Figure SAcknowledgmentsWe are grateful to Min-Ji Song at Yonsei Medical Research Center for technical assistance.Author ContributionsConceived and designed the experiments: Hyojin Kim M-SK GW Hyongbum Kim J-SK. Performed the experiments: Hyojin Kim M-SK 25033180 GW CL. Analyzed the data: Hyojin Kim M-SK GW Hyongbum Kim JSK. Wrote the paper: Hyongbum Kim J-SK.
Avian B cell development occurs in the bursa of Fabricius, a specialized organ with a follicular structure of mesenchymal and epithelial elements [1]. The primary role of the bursa of Fabricius, officially called the bursa cloacalis, is to function as the central locus of B cell development [2]. Bursal follicular development is induced by immigration of lymphoid stem cells, some of splenic origin, through mesenchyme to reach the bursal epithelium where they undergo proliferative expansion [3]. The Bu-1 antigen is one of the earliest antigens to appear in B cell ontogeny and is expressed on virtually all B cells except for plasma cells [4,5]. It thus serves as a marker for stage of maturation of B cells. Siatskas and Boyd [6] reported that the Bu-1 antigen is a marker which 374913-63-0 site identifies B-cell precursors with pre-bursal stem cell activity. B cell development in the bursa of Fabricius is dependent on the presence of an epithelial reticulum [7]. Consistent with this, bursal epithelium has been reported to be necessary if immigrant stem cells are to proliferate and commence gene conversion [3]. The bursa is a target for several avian viruses including infectious bursal disease virus (IBDV), Marek’s disease and Newcastle Disease virus (NDV) [8,9]. In the case of IBDV, studies have shown that the target cells are the B lymphocytes and virus replication leads to extensive bursal Rubusoside biological activity follicle destruction [10]. Whereas in vivo systems are mandatory for studying migratory patterns within the lymphoid system, in vitro systems which permit continuous observation and experimental manipulation cancomplement understanding of both normal development and host responses to pathogens. Although in vitro analogues of mammalian B cell development exist [11,12] comparable avian systems are lacking even though B cell development in the chick bursa was described before extrapolation of this observation to mammals. This study aimed to develop 1317923 an in vitro system which mimics avian bursal B cell development to permit investigation of the response to relevant pathogens.Materials and Methods Ethics StatementAll experiments were conducted in accord with the guidelines of laboratory animal care of Universiti Putra Malaysia, (Ref: UPM Research Policy). Approval of the ethics committee is not needed for work carried out in chicken embryos before the time of hatching, (Ref: UPM/FPV/PU/B901).Agglomerate CultureEmbryos used were from an outbred broiler strain and all fertilized eggs were incubated at 38uC. Single cell suspensions were prepared from spleen and a mixture of proventriculus and intestine from the same individual 15?0 day chick embryos (to avoid any confounding effects of allogeneic interactions) by enzymatic disaggregation. Briefly, proventriculus and intestine were minced and incubated in 2000 Unit/ml collagenase (Sigma, USA) in PBS supplemented with 0.1 BSA and 0.Ng ZFN (Z891) were cotransfected into HEK293 cells, hygromycin selection was performed by culturing the cells in the presence of 2 mg/ml hygromycin B for two days. The selected or unselected (control) cells were plated at a density of 3,000 cells/100 mm dish,Figure SAcknowledgmentsWe are grateful to Min-Ji Song at Yonsei Medical Research Center for technical assistance.Author ContributionsConceived and designed the experiments: Hyojin Kim M-SK GW Hyongbum Kim J-SK. Performed the experiments: Hyojin Kim M-SK 25033180 GW CL. Analyzed the data: Hyojin Kim M-SK GW Hyongbum Kim JSK. Wrote the paper: Hyongbum Kim J-SK.
Avian B cell development occurs in the bursa of Fabricius, a specialized organ with a follicular structure of mesenchymal and epithelial elements [1]. The primary role of the bursa of Fabricius, officially called the bursa cloacalis, is to function as the central locus of B cell development [2]. Bursal follicular development is induced by immigration of lymphoid stem cells, some of splenic origin, through mesenchyme to reach the bursal epithelium where they undergo proliferative expansion [3]. The Bu-1 antigen is one of the earliest antigens to appear in B cell ontogeny and is expressed on virtually all B cells except for plasma cells [4,5]. It thus serves as a marker for stage of maturation of B cells. Siatskas and Boyd [6] reported that the Bu-1 antigen is a marker which identifies B-cell precursors with pre-bursal stem cell activity. B cell development in the bursa of Fabricius is dependent on the presence of an epithelial reticulum [7]. Consistent with this, bursal epithelium has been reported to be necessary if immigrant stem cells are to proliferate and commence gene conversion [3]. The bursa is a target for several avian viruses including infectious bursal disease virus (IBDV), Marek’s disease and Newcastle Disease virus (NDV) [8,9]. In the case of IBDV, studies have shown that the target cells are the B lymphocytes and virus replication leads to extensive bursal follicle destruction [10]. Whereas in vivo systems are mandatory for studying migratory patterns within the lymphoid system, in vitro systems which permit continuous observation and experimental manipulation cancomplement understanding of both normal development and host responses to pathogens. Although in vitro analogues of mammalian B cell development exist [11,12] comparable avian systems are lacking even though B cell development in the chick bursa was described before extrapolation of this observation to mammals. This study aimed to develop 1317923 an in vitro system which mimics avian bursal B cell development to permit investigation of the response to relevant pathogens.Materials and Methods Ethics StatementAll experiments were conducted in accord with the guidelines of laboratory animal care of Universiti Putra Malaysia, (Ref: UPM Research Policy). Approval of the ethics committee is not needed for work carried out in chicken embryos before the time of hatching, (Ref: UPM/FPV/PU/B901).Agglomerate CultureEmbryos used were from an outbred broiler strain and all fertilized eggs were incubated at 38uC. Single cell suspensions were prepared from spleen and a mixture of proventriculus and intestine from the same individual 15?0 day chick embryos (to avoid any confounding effects of allogeneic interactions) by enzymatic disaggregation. Briefly, proventriculus and intestine were minced and incubated in 2000 Unit/ml collagenase (Sigma, USA) in PBS supplemented with 0.1 BSA and 0.
