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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.


Related in: MedlinePlus

Absence of D antigen-mediated agglutination in RHD-knockout cell lines.Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to an agglutination test using anti-D blood grouping reagents (a) and a weak D test using anti-D blood grouping reagents and Coombs' reagent (Anti-IgG, -C3d) (b) in 96-well plates and on glass slides. Rh D-positive, D-negative and weak D-positive human peripheral blood cells were used as the controls. A photograph and photomicrographs of each cell line are shown. Scale bar, 500 μm.
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f6: Absence of D antigen-mediated agglutination in RHD-knockout cell lines.Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to an agglutination test using anti-D blood grouping reagents (a) and a weak D test using anti-D blood grouping reagents and Coombs' reagent (Anti-IgG, -C3d) (b) in 96-well plates and on glass slides. Rh D-positive, D-negative and weak D-positive human peripheral blood cells were used as the controls. A photograph and photomicrographs of each cell line are shown. Scale bar, 500 μm.

Mentions: We next performed an agglutination test, which is used for blood group typing. Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to the agglutination test using anti-D blood grouping reagents. The mixture of cells and anti-D reagent was incubated for 15 min at 37 °C, shaken with pipet tips and observed in 96-well plates and on glass slides. As positive and negative controls, we used Rh D-positive and -negative blood cells, which showed agglutination and no agglutination, respectively (Fig. 6a, Supplementary Fig. 9). Parental and E4_M, but not E1_B and E4_B, showed agglutination, suggesting that E1_B and E4_B are phenotypically Rh D-negative.


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)

Absence of D antigen-mediated agglutination in RHD-knockout cell lines.Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to an agglutination test using anti-D blood grouping reagents (a) and a weak D test using anti-D blood grouping reagents and Coombs' reagent (Anti-IgG, -C3d) (b) in 96-well plates and on glass slides. Rh D-positive, D-negative and weak D-positive human peripheral blood cells were used as the controls. A photograph and photomicrographs of each cell line are shown. Scale bar, 500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Absence of D antigen-mediated agglutination in RHD-knockout cell lines.Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to an agglutination test using anti-D blood grouping reagents (a) and a weak D test using anti-D blood grouping reagents and Coombs' reagent (Anti-IgG, -C3d) (b) in 96-well plates and on glass slides. Rh D-positive, D-negative and weak D-positive human peripheral blood cells were used as the controls. A photograph and photomicrographs of each cell line are shown. Scale bar, 500 μm.
Mentions: We next performed an agglutination test, which is used for blood group typing. Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to the agglutination test using anti-D blood grouping reagents. The mixture of cells and anti-D reagent was incubated for 15 min at 37 °C, shaken with pipet tips and observed in 96-well plates and on glass slides. As positive and negative controls, we used Rh D-positive and -negative blood cells, which showed agglutination and no agglutination, respectively (Fig. 6a, Supplementary Fig. 9). Parental and E4_M, but not E1_B and E4_B, showed agglutination, suggesting that E1_B and E4_B are phenotypically Rh D-negative.

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.


Related in: MedlinePlus