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Rh D blood group conversion using transcription activator-like effector nucleases.

Kim YH, Kim HO, Baek EJ, Kurita R, Cha HJ, Nakamura Y, Kim H - Nat Commun (2015)

Bottom Line: Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs).After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained.Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, South Korea [2] Department of Pharmacology, Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Yonsei University College of Medicine, Seoul 120-752, South Korea.

ABSTRACT
Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

No MeSH data available.


Generation of RHD-mutated erythroid progenitor cells.(a) Schematic representation illustrating the process of RHD-mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD. Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. (b) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers
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f2: Generation of RHD-mutated erythroid progenitor cells.(a) Schematic representation illustrating the process of RHD-mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD. Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. (b) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers

Mentions: We next attempted to use these TALENs to make RHD-knockout cells from Rh D-positive erythroid progenitor cells. Our source of erythroid progenitor cells was the HiDEP-1 cell line, which is derived from induced pluripotent stem cells generated from fibroblasts from an Rh D-positive (DD) donor19. For efficient generation of knockout cells, we used a magnetic reporter plasmid that expresses H-2Kk in the presence of programmable nuclease activity at the target sequence as previously reported151720 (Supplementary Fig. 2). Three days after co-transfection with the reporter plasmid and plasmids encoding RHD_E1_TALENs or RHD_E4_TALENs, H-2Kk+ HiDEP-1 cells were magnetically separated1520 and seeded into 96-well plates for dilution cloning at an average density of 0.25 cells per well (Fig. 2a). Individual clones were isolated 17 days after the clonal cell seeding; genomic DNA was isolated from each clone and analysed. For efficient screening of mutant clones, genomic DNA from three (RHD_E1_TALENs) or four (RHD_E4_TALENs) clones was grouped, mixed and subjected to the T7E1 assay. This group T7E1 assay showed that six out of seven clone groups from the RHD_E1_TALEN-transfected cells and nine out of thirty-seven clone groups from the RHD_E4_TALEN-transfected cells included at least one mutant clone (Supplementary Fig. 3). Each individual clone in these positive clone groups was then subjected to the T7E1 assay. In cells transfected with RHD_E1_TALENs, this screening identified 9 mutant clones out of 20 total (Supplementary Fig. 3; Fig. 2b). Subsequent sequencing analysis showed that these nine clones comprise seven monoallelic out-of-frame mutant clones, 1 monoallelic in-frame mutant clone (3 nucleotide deletion), and 1 biallelic mutant clone that contains one in-frame mutation (6 nucleotide deletion) and one out-of-frame mutation (2 nucleotide deletion)(Supplementary Fig. 4a). Because in-frame mutations can lead to incomplete gene knockout, we attempted to obtain biallelic out-of-frame mutant clones. For this purpose, we again co-transfected the reporter plasmid and the plasmids encoding RHD_E1_TALENs into one (#17) of the 7 monoallelic out-of-frame mutant clones and performed clonal culture after magnetic separation. Sequencing of DNA from 7 subclones derived from this monoallelic mutant clone showed that all the subclones contain the 2 nucleotide deletion that was initially observed in the parent clone. Among the 7 subclones, one subclone (#17-3), which contained biallelic out-of-frame mutations, was designated as E1_B (#17-3; Supplementary Fig. 4b) and expanded for additional study.


Rh D blood group conversion using transcription activator-like effector nucleases.

Kim YH, Kim HO, Baek EJ, Kurita R, Cha HJ, Nakamura Y, Kim H - Nat Commun (2015)

Generation of RHD-mutated erythroid progenitor cells.(a) Schematic representation illustrating the process of RHD-mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD. Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. (b) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490371&req=5

f2: Generation of RHD-mutated erythroid progenitor cells.(a) Schematic representation illustrating the process of RHD-mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD. Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. (b) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers
Mentions: We next attempted to use these TALENs to make RHD-knockout cells from Rh D-positive erythroid progenitor cells. Our source of erythroid progenitor cells was the HiDEP-1 cell line, which is derived from induced pluripotent stem cells generated from fibroblasts from an Rh D-positive (DD) donor19. For efficient generation of knockout cells, we used a magnetic reporter plasmid that expresses H-2Kk in the presence of programmable nuclease activity at the target sequence as previously reported151720 (Supplementary Fig. 2). Three days after co-transfection with the reporter plasmid and plasmids encoding RHD_E1_TALENs or RHD_E4_TALENs, H-2Kk+ HiDEP-1 cells were magnetically separated1520 and seeded into 96-well plates for dilution cloning at an average density of 0.25 cells per well (Fig. 2a). Individual clones were isolated 17 days after the clonal cell seeding; genomic DNA was isolated from each clone and analysed. For efficient screening of mutant clones, genomic DNA from three (RHD_E1_TALENs) or four (RHD_E4_TALENs) clones was grouped, mixed and subjected to the T7E1 assay. This group T7E1 assay showed that six out of seven clone groups from the RHD_E1_TALEN-transfected cells and nine out of thirty-seven clone groups from the RHD_E4_TALEN-transfected cells included at least one mutant clone (Supplementary Fig. 3). Each individual clone in these positive clone groups was then subjected to the T7E1 assay. In cells transfected with RHD_E1_TALENs, this screening identified 9 mutant clones out of 20 total (Supplementary Fig. 3; Fig. 2b). Subsequent sequencing analysis showed that these nine clones comprise seven monoallelic out-of-frame mutant clones, 1 monoallelic in-frame mutant clone (3 nucleotide deletion), and 1 biallelic mutant clone that contains one in-frame mutation (6 nucleotide deletion) and one out-of-frame mutation (2 nucleotide deletion)(Supplementary Fig. 4a). Because in-frame mutations can lead to incomplete gene knockout, we attempted to obtain biallelic out-of-frame mutant clones. For this purpose, we again co-transfected the reporter plasmid and the plasmids encoding RHD_E1_TALENs into one (#17) of the 7 monoallelic out-of-frame mutant clones and performed clonal culture after magnetic separation. Sequencing of DNA from 7 subclones derived from this monoallelic mutant clone showed that all the subclones contain the 2 nucleotide deletion that was initially observed in the parent clone. Among the 7 subclones, one subclone (#17-3), which contained biallelic out-of-frame mutations, was designated as E1_B (#17-3; Supplementary Fig. 4b) and expanded for additional study.

Bottom Line: Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs).After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained.Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, South Korea [2] Department of Pharmacology, Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Yonsei University College of Medicine, Seoul 120-752, South Korea.

ABSTRACT
Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

No MeSH data available.