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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.


TALENs targeting the human RHD gene.(a) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD_E1_TALENs and RHD_E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. (b,c) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 (b, RHD_E1_TALENs) or exon 4 (c, RHD_E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.
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f1: TALENs targeting the human RHD gene.(a) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD_E1_TALENs and RHD_E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. (b,c) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 (b, RHD_E1_TALENs) or exon 4 (c, RHD_E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.

Mentions: To completely disrupt the RHD gene, we first obtained a TALEN pair that targets upstream of the protein-coding region; a TALEN pair targeting exon 1 was prepared (RHD_E1_TALENs; Fig. 1a). We next determined that this exon 1 target sequence is included in all transcript variants. Transcript information from NCBI and Ensembl showed that the human RHD gene has collectively 10 transcript variants including two that do not produce proteins (Supplementary Fig. 1). Exon 4 is included in all eight coding sequences, whereas exon 1 is included in seven coding sequences. Furthermore, exon 4 is the mutation locus of RHD in some Rh D-negative people11. Thus, we also designed TALENs that target exon 4 (RHD_E4_TALENs; Fig. 1a).


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)

TALENs targeting the human RHD gene.(a) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD_E1_TALENs and RHD_E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. (b,c) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 (b, RHD_E1_TALENs) or exon 4 (c, RHD_E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: TALENs targeting the human RHD gene.(a) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD_E1_TALENs and RHD_E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. (b,c) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 (b, RHD_E1_TALENs) or exon 4 (c, RHD_E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.
Mentions: To completely disrupt the RHD gene, we first obtained a TALEN pair that targets upstream of the protein-coding region; a TALEN pair targeting exon 1 was prepared (RHD_E1_TALENs; Fig. 1a). We next determined that this exon 1 target sequence is included in all transcript variants. Transcript information from NCBI and Ensembl showed that the human RHD gene has collectively 10 transcript variants including two that do not produce proteins (Supplementary Fig. 1). Exon 4 is included in all eight coding sequences, whereas exon 1 is included in seven coding sequences. Furthermore, exon 4 is the mutation locus of RHD in some Rh D-negative people11. Thus, we also designed TALENs that target exon 4 (RHD_E4_TALENs; Fig. 1a).

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.