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Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

Epsztejn-Litman S, Cohen-Hadad Y, Aharoni S, Altarescu G, Renbaum P, Levy-Lahad E, Schonberger O, Eldar-Geva T, Zeligson S, Eiges R - PLoS ONE (2015)

Bottom Line: By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome.Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation.As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Shaare Zedek Medical Center affiliated with the Hebrew University School of Medicine, Jerusalem, Israel.

ABSTRACT
We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

No MeSH data available.


Related in: MedlinePlus

SMN1 genotyping and haplotype analysis in SZ-SMA5 HESCs.Homozygous loss of SMN1 was assessed by (A) PCR amplification of exon 7 with a mismatch forward primer that creates a DraI restriction site in SMN2, but not in SMN1. After DraI restriction digestion, wild-type samples exhibit two bands in this assay while samples completely lacking SMN1 exhibit only one band. Depicted are two wild-type (WT1 and WT2) HESC lines; SZ-SMA5; and an unrelated SMA-affected HESC line with homozygous deletion of SMN1 (SZ-SMA6) as a positive control. (B) SMN1 and SMN2 copy number was assayed with a commercial MLPA test. Capillary electrophoresis patterns of DNA samples from normal (wild-type) and SZ-SMA5 cells illustrates homozygous deletion of SMN1 as indicated by the gene-specific absence of PCR products from exons 7 (183 nt) and 8 (218 nt) (marked by red arrows). (C) PCR fragment sizes of SMN1-flanking microsatellites in SZ-SMA5 demonstrate that the HESCs were derived from an embryo which inherited a maternal mutation-linked allele, but no paternal allele.
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pone.0138893.g002: SMN1 genotyping and haplotype analysis in SZ-SMA5 HESCs.Homozygous loss of SMN1 was assessed by (A) PCR amplification of exon 7 with a mismatch forward primer that creates a DraI restriction site in SMN2, but not in SMN1. After DraI restriction digestion, wild-type samples exhibit two bands in this assay while samples completely lacking SMN1 exhibit only one band. Depicted are two wild-type (WT1 and WT2) HESC lines; SZ-SMA5; and an unrelated SMA-affected HESC line with homozygous deletion of SMN1 (SZ-SMA6) as a positive control. (B) SMN1 and SMN2 copy number was assayed with a commercial MLPA test. Capillary electrophoresis patterns of DNA samples from normal (wild-type) and SZ-SMA5 cells illustrates homozygous deletion of SMN1 as indicated by the gene-specific absence of PCR products from exons 7 (183 nt) and 8 (218 nt) (marked by red arrows). (C) PCR fragment sizes of SMN1-flanking microsatellites in SZ-SMA5 demonstrate that the HESCs were derived from an embryo which inherited a maternal mutation-linked allele, but no paternal allele.

Mentions: The high similarity between SMN1 and SMN2 complicates direct genotyping for SMA diagnosis. To overcome this problem and verify homozygote deletion of SMN1 in SZ-SMA5, we utilized two established assays. In one assay we PCR amplified exon 7 with a mismatch forward primer that differentially creates a DraI restriction site in SMN2, but not SMN1 [21] such that all exon 7 molecules will be cleaved in the absence of SMN1 (unlike for homozygote wild type or heterozygote samples in which SMN1 exon 7 molecules will not be cleaved). In the second assay, we assessed copy number variations in SMN1 and SMN2 with a commercial multiplex ligation-dependent probe amplification (MLPA) kit featuring sequence-specific probes that differentiate between copy number changes in SMN1 and SMN2. Using both diagnostic procedures we confirmed the PGD test results for SZ-SMA5 by demonstrating the SMN1-specific absence of both exons 7 and 8 (Fig 2A and 2B). In addition, we validated the transmission of parental mutant chromosomes to SZ-SMA5 by carrying out haplotype analysis using 5 gene-flanking informative microsatellite markers for embryo typing. Interestingly, this allele-specific confirmation identified only the mother’s mutant SMN1 allele. No paternal alleles (wild type or mutant) were identified with the same markers (Fig 2C).


Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

Epsztejn-Litman S, Cohen-Hadad Y, Aharoni S, Altarescu G, Renbaum P, Levy-Lahad E, Schonberger O, Eldar-Geva T, Zeligson S, Eiges R - PLoS ONE (2015)

SMN1 genotyping and haplotype analysis in SZ-SMA5 HESCs.Homozygous loss of SMN1 was assessed by (A) PCR amplification of exon 7 with a mismatch forward primer that creates a DraI restriction site in SMN2, but not in SMN1. After DraI restriction digestion, wild-type samples exhibit two bands in this assay while samples completely lacking SMN1 exhibit only one band. Depicted are two wild-type (WT1 and WT2) HESC lines; SZ-SMA5; and an unrelated SMA-affected HESC line with homozygous deletion of SMN1 (SZ-SMA6) as a positive control. (B) SMN1 and SMN2 copy number was assayed with a commercial MLPA test. Capillary electrophoresis patterns of DNA samples from normal (wild-type) and SZ-SMA5 cells illustrates homozygous deletion of SMN1 as indicated by the gene-specific absence of PCR products from exons 7 (183 nt) and 8 (218 nt) (marked by red arrows). (C) PCR fragment sizes of SMN1-flanking microsatellites in SZ-SMA5 demonstrate that the HESCs were derived from an embryo which inherited a maternal mutation-linked allele, but no paternal allele.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608834&req=5

pone.0138893.g002: SMN1 genotyping and haplotype analysis in SZ-SMA5 HESCs.Homozygous loss of SMN1 was assessed by (A) PCR amplification of exon 7 with a mismatch forward primer that creates a DraI restriction site in SMN2, but not in SMN1. After DraI restriction digestion, wild-type samples exhibit two bands in this assay while samples completely lacking SMN1 exhibit only one band. Depicted are two wild-type (WT1 and WT2) HESC lines; SZ-SMA5; and an unrelated SMA-affected HESC line with homozygous deletion of SMN1 (SZ-SMA6) as a positive control. (B) SMN1 and SMN2 copy number was assayed with a commercial MLPA test. Capillary electrophoresis patterns of DNA samples from normal (wild-type) and SZ-SMA5 cells illustrates homozygous deletion of SMN1 as indicated by the gene-specific absence of PCR products from exons 7 (183 nt) and 8 (218 nt) (marked by red arrows). (C) PCR fragment sizes of SMN1-flanking microsatellites in SZ-SMA5 demonstrate that the HESCs were derived from an embryo which inherited a maternal mutation-linked allele, but no paternal allele.
Mentions: The high similarity between SMN1 and SMN2 complicates direct genotyping for SMA diagnosis. To overcome this problem and verify homozygote deletion of SMN1 in SZ-SMA5, we utilized two established assays. In one assay we PCR amplified exon 7 with a mismatch forward primer that differentially creates a DraI restriction site in SMN2, but not SMN1 [21] such that all exon 7 molecules will be cleaved in the absence of SMN1 (unlike for homozygote wild type or heterozygote samples in which SMN1 exon 7 molecules will not be cleaved). In the second assay, we assessed copy number variations in SMN1 and SMN2 with a commercial multiplex ligation-dependent probe amplification (MLPA) kit featuring sequence-specific probes that differentiate between copy number changes in SMN1 and SMN2. Using both diagnostic procedures we confirmed the PGD test results for SZ-SMA5 by demonstrating the SMN1-specific absence of both exons 7 and 8 (Fig 2A and 2B). In addition, we validated the transmission of parental mutant chromosomes to SZ-SMA5 by carrying out haplotype analysis using 5 gene-flanking informative microsatellite markers for embryo typing. Interestingly, this allele-specific confirmation identified only the mother’s mutant SMN1 allele. No paternal alleles (wild type or mutant) were identified with the same markers (Fig 2C).

Bottom Line: By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome.Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation.As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Shaare Zedek Medical Center affiliated with the Hebrew University School of Medicine, Jerusalem, Israel.

ABSTRACT
We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

No MeSH data available.


Related in: MedlinePlus