Limits...
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.


DNA sequences of RHD-mutated clones.The RHD gene DNA sequences from the parental cells, clones with biallelic mutations in exon 1 (E1_B; a) or exon 4 (E4_B; b), and a clone with a monoallelic mutation in exon 4 (E4_M; c). TALE-binding sites are in a red font and spacer regions are indicated with green boxes. Deleted bases are indicated by dashes and inserted bases are shown in a blue font. The number of occurrences is shown in parentheses (for example, × 7 and × 5 indicate the number of each sequence). The sequence and sequencing chromatogram for each allele are shown. The locus of each mutation, the PTC generated by the mutation and the distance between the PTC and the exon–intron junction are depicted in a schematic of the RHD gene. Expected protein sequence translated from each allele are displayed, such that mutated protein sequences generated by a nuclease-induced frameshifting mutation are shown in a red font and translation termination is indicated with a dash. nt, nucleotide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490371&req=5

f3: DNA sequences of RHD-mutated clones.The RHD gene DNA sequences from the parental cells, clones with biallelic mutations in exon 1 (E1_B; a) or exon 4 (E4_B; b), and a clone with a monoallelic mutation in exon 4 (E4_M; c). TALE-binding sites are in a red font and spacer regions are indicated with green boxes. Deleted bases are indicated by dashes and inserted bases are shown in a blue font. The number of occurrences is shown in parentheses (for example, × 7 and × 5 indicate the number of each sequence). The sequence and sequencing chromatogram for each allele are shown. The locus of each mutation, the PTC generated by the mutation and the distance between the PTC and the exon–intron junction are depicted in a schematic of the RHD gene. Expected protein sequence translated from each allele are displayed, such that mutated protein sequences generated by a nuclease-induced frameshifting mutation are shown in a red font and translation termination is indicated with a dash. nt, nucleotide.

Mentions: We next examined the sequencing results to determine whether the selected mutant clones have PTCs in RHD exons. Both RHD alleles of clone E1_B had PTCs within exon 1 (Fig. 3a). In clone E4_B, PTCs were observed in exon 4 of one allele and in exon 5 of the other (Fig. 3b). In clone E4_M, a PTC was in exon 4 of one mutated allele (Fig. 3c). We have indicated the locations of the PTCs in a two-dimensional (2D) model of the RHD protein in Supplementary Fig. 5. Taken together, these results indicate that the RHD protein will be expressed in clone E4_M but not in E1_B or E4_B.


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)

DNA sequences of RHD-mutated clones.The RHD gene DNA sequences from the parental cells, clones with biallelic mutations in exon 1 (E1_B; a) or exon 4 (E4_B; b), and a clone with a monoallelic mutation in exon 4 (E4_M; c). TALE-binding sites are in a red font and spacer regions are indicated with green boxes. Deleted bases are indicated by dashes and inserted bases are shown in a blue font. The number of occurrences is shown in parentheses (for example, × 7 and × 5 indicate the number of each sequence). The sequence and sequencing chromatogram for each allele are shown. The locus of each mutation, the PTC generated by the mutation and the distance between the PTC and the exon–intron junction are depicted in a schematic of the RHD gene. Expected protein sequence translated from each allele are displayed, such that mutated protein sequences generated by a nuclease-induced frameshifting mutation are shown in a red font and translation termination is indicated with a dash. nt, nucleotide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: DNA sequences of RHD-mutated clones.The RHD gene DNA sequences from the parental cells, clones with biallelic mutations in exon 1 (E1_B; a) or exon 4 (E4_B; b), and a clone with a monoallelic mutation in exon 4 (E4_M; c). TALE-binding sites are in a red font and spacer regions are indicated with green boxes. Deleted bases are indicated by dashes and inserted bases are shown in a blue font. The number of occurrences is shown in parentheses (for example, × 7 and × 5 indicate the number of each sequence). The sequence and sequencing chromatogram for each allele are shown. The locus of each mutation, the PTC generated by the mutation and the distance between the PTC and the exon–intron junction are depicted in a schematic of the RHD gene. Expected protein sequence translated from each allele are displayed, such that mutated protein sequences generated by a nuclease-induced frameshifting mutation are shown in a red font and translation termination is indicated with a dash. nt, nucleotide.
Mentions: We next examined the sequencing results to determine whether the selected mutant clones have PTCs in RHD exons. Both RHD alleles of clone E1_B had PTCs within exon 1 (Fig. 3a). In clone E4_B, PTCs were observed in exon 4 of one allele and in exon 5 of the other (Fig. 3b). In clone E4_M, a PTC was in exon 4 of one mutated allele (Fig. 3c). We have indicated the locations of the PTCs in a two-dimensional (2D) model of the RHD protein in Supplementary Fig. 5. Taken together, these results indicate that the RHD protein will be expressed in clone E4_M but not in E1_B or E4_B.

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.