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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.


Flow cytometric analysis of D antigen expression in mutated cells.Parental and RHD-mutated (biallelic, E1_B, E4_B; monoallelic, E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to flow cytometry. D antigen expression was determined in glycophorin A+ cells. (a) Representative histograms. (b) The percentage of D antigen-positive cells in the population of glycophorin A-positive cells. ANOVA followed with Bonferroni's multiple comparison was performed (***P<0.001, **P<0.01, ns=not significant; n=3). Error bars represent the s.e.m.
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f5: Flow cytometric analysis of D antigen expression in mutated cells.Parental and RHD-mutated (biallelic, E1_B, E4_B; monoallelic, E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to flow cytometry. D antigen expression was determined in glycophorin A+ cells. (a) Representative histograms. (b) The percentage of D antigen-positive cells in the population of glycophorin A-positive cells. ANOVA followed with Bonferroni's multiple comparison was performed (***P<0.001, **P<0.01, ns=not significant; n=3). Error bars represent the s.e.m.

Mentions: We next evaluated D antigen protein expression on each HiDEP-1 cell clone using flow cytometry. As positive and negative controls for this analysis, we used peripheral blood cells isolated from Rh D-positive and -negative donors, respectively. Among glycophorin A+ cells, the D antigen expression rate was 99.96±0.004% and 1.4±0.6% in Rh D-positive and -negative blood cells, respectively (Fig. 5), suggesting that flow cytometry is a sensitive way to detect D antigen expression with an ∼1% nonspecific background signal.


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)

Flow cytometric analysis of D antigen expression in mutated cells.Parental and RHD-mutated (biallelic, E1_B, E4_B; monoallelic, E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to flow cytometry. D antigen expression was determined in glycophorin A+ cells. (a) Representative histograms. (b) The percentage of D antigen-positive cells in the population of glycophorin A-positive cells. ANOVA followed with Bonferroni's multiple comparison was performed (***P<0.001, **P<0.01, ns=not significant; n=3). Error bars represent the s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Flow cytometric analysis of D antigen expression in mutated cells.Parental and RHD-mutated (biallelic, E1_B, E4_B; monoallelic, E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to flow cytometry. D antigen expression was determined in glycophorin A+ cells. (a) Representative histograms. (b) The percentage of D antigen-positive cells in the population of glycophorin A-positive cells. ANOVA followed with Bonferroni's multiple comparison was performed (***P<0.001, **P<0.01, ns=not significant; n=3). Error bars represent the s.e.m.
Mentions: We next evaluated D antigen protein expression on each HiDEP-1 cell clone using flow cytometry. As positive and negative controls for this analysis, we used peripheral blood cells isolated from Rh D-positive and -negative donors, respectively. Among glycophorin A+ cells, the D antigen expression rate was 99.96±0.004% and 1.4±0.6% in Rh D-positive and -negative blood cells, respectively (Fig. 5), suggesting that flow cytometry is a sensitive way to detect D antigen expression with an ∼1% nonspecific background signal.

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.