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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.


RHD mRNA in the mutated clones.RT–PCR was performed to detect RHD mRNA in each clone and the amplicons were subjected to electrophoresis (a) and sequencing (b). (a) Representative pictures of electrophoresis. ACTB was used as control. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). (b) Schematic representation of RHD mRNA sequences. The number of occurrences is shown on the right of each transcript. Blue and red circles indicate normal and mutated exons, respectively. For some amplicons, the sequence and sequencing chromatogram are shown (spacer regions are indicated with green boxes).
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f4: RHD mRNA in the mutated clones.RT–PCR was performed to detect RHD mRNA in each clone and the amplicons were subjected to electrophoresis (a) and sequencing (b). (a) Representative pictures of electrophoresis. ACTB was used as control. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). (b) Schematic representation of RHD mRNA sequences. The number of occurrences is shown on the right of each transcript. Blue and red circles indicate normal and mutated exons, respectively. For some amplicons, the sequence and sequencing chromatogram are shown (spacer regions are indicated with green boxes).

Mentions: We next examined RHD mRNA expression in parental, E1_B, E4_B and E4_M HiDEP-1 cells using RT–PCR. Electrophoresis of the RT–PCR products from parental HiDEP-1 cells showed a dense band at ∼1.4 kb, a medium-density band at ∼1.3 kb and a weak band at ∼1.1 kb (Fig. 4a). The two higher molecular weight bands were also clearly observed in E1_B and E4_M samples as well. To identify these bands, we cloned the RT–PCR products into T-vectors and performed capillary sequencing. The results showed that transcript variant 1, which lacks exon 8, and a new transcript variant 1, which lacks both exons 7 and 8, were observed at frequencies of 8/19 and 4/19 in parental cells, 6/15 and 4/15 in E1_B and 10/20 and 8/20 in E4_M, respectively, accounting for the two most frequently observed transcript variants in parental (8/19+4/19=63%), E1_B (6/15+4/15=67%) and E4_B (10/20+8/20=90%; Fig. 4b). Furthermore, the expected sizes of variant 1 and the new variant 1 are 1,422 and 1,288 bp, respectively. Taken together, these results indicate that the dense band at 1.4 kb and the medium-density band at 1.3 kb are variant 1 and new variant 1, respectively, and suggest that these two transcript variants are dominant in HiDEP-1 cells.


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)

RHD mRNA in the mutated clones.RT–PCR was performed to detect RHD mRNA in each clone and the amplicons were subjected to electrophoresis (a) and sequencing (b). (a) Representative pictures of electrophoresis. ACTB was used as control. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). (b) Schematic representation of RHD mRNA sequences. The number of occurrences is shown on the right of each transcript. Blue and red circles indicate normal and mutated exons, respectively. For some amplicons, the sequence and sequencing chromatogram are shown (spacer regions are indicated with green boxes).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: RHD mRNA in the mutated clones.RT–PCR was performed to detect RHD mRNA in each clone and the amplicons were subjected to electrophoresis (a) and sequencing (b). (a) Representative pictures of electrophoresis. ACTB was used as control. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). (b) Schematic representation of RHD mRNA sequences. The number of occurrences is shown on the right of each transcript. Blue and red circles indicate normal and mutated exons, respectively. For some amplicons, the sequence and sequencing chromatogram are shown (spacer regions are indicated with green boxes).
Mentions: We next examined RHD mRNA expression in parental, E1_B, E4_B and E4_M HiDEP-1 cells using RT–PCR. Electrophoresis of the RT–PCR products from parental HiDEP-1 cells showed a dense band at ∼1.4 kb, a medium-density band at ∼1.3 kb and a weak band at ∼1.1 kb (Fig. 4a). The two higher molecular weight bands were also clearly observed in E1_B and E4_M samples as well. To identify these bands, we cloned the RT–PCR products into T-vectors and performed capillary sequencing. The results showed that transcript variant 1, which lacks exon 8, and a new transcript variant 1, which lacks both exons 7 and 8, were observed at frequencies of 8/19 and 4/19 in parental cells, 6/15 and 4/15 in E1_B and 10/20 and 8/20 in E4_M, respectively, accounting for the two most frequently observed transcript variants in parental (8/19+4/19=63%), E1_B (6/15+4/15=67%) and E4_B (10/20+8/20=90%; Fig. 4b). Furthermore, the expected sizes of variant 1 and the new variant 1 are 1,422 and 1,288 bp, respectively. Taken together, these results indicate that the dense band at 1.4 kb and the medium-density band at 1.3 kb are variant 1 and new variant 1, respectively, and suggest that these two transcript variants are dominant in HiDEP-1 cells.

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.