Uses of banding techniques for the identification of human diseases of cytogenetic origin.

Shaw, Margery W.

doi:10.1289/ehp.7306151

Cited by 3 publications

(2 citation statements)

References 29 publications

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“…Potential off-targets (score ≤ 0.9 according to design tool; http://crispr.mit.edu/ ; Hsu et al., 2013 ) were tested by Sanger sequencing of corresponding PCR products that were amplified from genomic DNA using specific primers for the iNGN cell line ( Table S4 ). 20 metaphase states from each cell line were karyotyped using G-banding ( Shaw, 1973 ) via the iPS facility of the Center for Molecular and Cellular Bioengineering (CMCB) at the Institute of Human Genetics, University Clinics Jena, Germany, to test for chromosomal alterations.…”

Section: Methodsmentioning

confidence: 99%

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis

et al. 2018

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SummaryNon-coding RNAs regulate many biological processes including neurogenesis. The brain-enriched miR-124 has been assigned as a key player of neuronal differentiation via its complex but little understood regulation of thousands of annotated targets. To systematically chart its regulatory functions, we used CRISPR/Cas9 gene editing to disrupt all six miR-124 alleles in human induced pluripotent stem cells. Upon neuronal induction, miR-124-deleted cells underwent neurogenesis and became functional neurons, albeit with altered morphology and neurotransmitter specification. Using RNA-induced-silencing-complex precipitation, we identified 98 high-confidence miR-124 targets, of which some directly led to decreased viability. By performing advanced transcription-factor-network analysis, we identified indirect miR-124 effects on apoptosis, neuronal subtype differentiation, and the regulation of previously uncharacterized zinc finger transcription factors. Our data emphasize the need for combined experimental- and system-level analyses to comprehensively disentangle and reveal miRNA functions, including their involvement in the neurogenesis of diverse neuronal cell types found in the human brain.

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Section: Methodsmentioning

confidence: 99%

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis

et al. 2018

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“…Single cells were harvested with TrypLE Express (Thermo Fisher Scientific, 12604013, Waltham, MA, USA), enlarged with 75 mM hypotonic KaryoMAX™ Potassium Chloride Solution (Thermo Fisher Scientific, 10575090, Waltham, MA, USA) for 20 min at 37 • C, fixed, and washed 3 times with 3:1 methanol:glacial acetic acid (Carl Roth, CP43.3, Karlsruhe, Germany and Merck, 1000631011, Darmstadt, Germany). Chromosome spreads were Giemsa stained and 20 metaphases of each sample were analyzed by G-banding [34] at the Institute of Human Genetics, University Clinics Jena, Germany.…”

Section: Reprogramming Hipsc Culture and Characterizationmentioning

confidence: 99%

Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

Sürün

Schneider

Mircetic

et al. 2020

Genes

101

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In contrast to CRISPR/Cas9 nucleases, CRISPR base editors (BE) and prime editors (PE) enable predefined nucleotide exchanges in genomic sequences without generating DNA double strand breaks. Here, we employed BE and PE mRNAs in conjunction with chemically synthesized sgRNAs and pegRNAs for efficient editing of human induced pluripotent stem cells (iPSC). Whereas we were unable to correct a disease-causing mutation in patient derived iPSCs using a CRISPR/Cas9 nuclease approach, we corrected the mutation back to wild type with high efficiency utilizing an adenine BE. We also used adenine and cytosine BEs to introduce nine different cancer associated TP53 mutations into human iPSCs with up to 90% efficiency, generating a panel of cell lines to investigate the biology of these mutations in an isogenic background. Finally, we pioneered the use of prime editing in human iPSCs, opening this important cell type for the precise modification of nucleotides not addressable by BEs and to multiple nucleotide exchanges. These approaches eliminate the necessity of deriving disease specific iPSCs from human donors and allows the comparison of different disease-causing mutations in isogenic genetic backgrounds.

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Banding technique used for the detection of chromosomal aberrations induced by radiation and alkylating agents TEPA and epichlorohydrin

Kuçerová

Polívková

1976

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Uses of banding techniques for the identification of human diseases of cytogenetic origin.

Cited by 3 publications

References 29 publications

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis

Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

Banding technique used for the detection of chromosomal aberrations induced by radiation and alkylating agents TEPA and epichlorohydrin

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