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Journal ArticleDOI

Isolation and Characterization of a Near-Haploid Human Cell Line

01 Nov 1999-Experimental Cell Research (Academic Press)-Vol. 252, Iss: 2, pp 273-280
TL;DR: By serial subcloning, a near-haploid subclone (P1-55) is isolated from a heterogeneous human leukemia cell line, KBM-7, which has approximately half the human diploid DNA content and has a haploid karyotype except for a disomy of chromosome 8.
About: This article is published in Experimental Cell Research.The article was published on 1999-11-01. It has received 136 citations till now. The article focuses on the topics: Karyotype & Ploidy.
Citations
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Journal ArticleDOI
TL;DR: The use of CRISPR-Cas9 technology has been instrumental in the characterization of the mutational landscape of complex human diseases like cancer, but despite the enormous rise in the identification of disease candidate genetic variants, their functionality is yet to be fully elucidated in order to have a clear implication in patient care as discussed by the authors .
Abstract: The use of next-generation sequencing (NGS) technologies has been instrumental in the characterization of the mutational landscape of complex human diseases like cancer. But despite the enormous rise in the identification of disease candidate genetic variants, their functionality is yet to be fully elucidated in order to have a clear implication in patient care. Haploid human cell models have become the tool of choice for functional gene studies, since they only contain one copy of the genome and can therefore show the unmasked phenotype of genetic variants. Over the past few years, the human near-haploid cell line HAP1 has widely been consolidated as one of the favorite cell line models for functional genetic studies. Its rapid turnover coupled with the fact that only one allele needs to be modified in order to express the subsequent desired phenotype has made this human cell line a valuable tool for gene editing by CRISPR-Cas9 technologies. This review examines the recent uses of the HAP1 cell line model in functional genetic studies and high-throughput genetic screens using the CRISPR-Cas9 system. It covers its use in an attempt to develop new and relevant disease models to further elucidate gene function, and create new ways to understand the genetic basis of human diseases. We will cover the advantages and potential of the use of CRISPR-Cas9 technology on HAP1 to easily and efficiently study the functional interpretation of gene function and human single-nucleotide genetic variants of unknown significance identified through NGS technologies, and its implications for changes in clinical practice and patient care.

1 citations

Journal ArticleDOI
20 Jun 2017
TL;DR: Interestingly, in mammalian systems, loss of function genetic approaches has lagged behind the gain of function approaches merely due to the scarcity of molecular tools that could function in high-throughput manner targeting both allele’s indiploid genomes.
Abstract: Viruses are lifeless, obligate, intracellular particles that serve as a carrier for their own genome, and require host cell components for replication. Through cell surface receptors these pathogens enter cells and adapt themselves with the host cellular machinery. Host cells, in turn, can neutralize the infection by sensing Pathogen-Associated Molecular Patterns (PAMPs), prompting the expression of antiviral proteins. The identification and characterization of these antiviral factors can provide ground-breaking insights into the host-virus alliance and basic facets of molecular biology leading towards discovery of potential targets in antiviral therapeutics. Approaches like forward genetic screening has markedly enhanced our understanding of fundamental biological processes in genetically tractable models like yeasts, drosophila, zebrafish and worms, however, these approaches were limited to these organisms. Due to the experimental complications, implicating forward genetics to cultured mammalian cells is a daunting and challenging task. Interestingly, in mammalian systems, loss of function genetic approaches has lagged behind the gain of function approaches merely due to the scarcity of molecular tools that could function in high-throughput manner targeting both allele’s indiploid genomes. Vector based ectopic expression of desired genes as a gain of function approach has been very successful in mammalian cells. Indeed, using this technique, various host cell surface receptors required for virus entry has been identified. Simply put, cell lines which are not susceptible for viral infection are transformed with cDNA libraries of viral infection permissive cell lines. The Hepatitis C viral entry through claudin1 and occluding receptors were identified using cDNA libraries from hepatocellular carcinoma cells. In contrast, loss of function approaches required stable knockout or knockdown of target loci. Initially RNAi approach has advanced our understanding of host-viral relationships [1,2]. Later on, a technique termed as haploid genetic screening had emerged which exploited the haploid human cell lines through insertion mutagenesis. Nearhaploid karyotype of chronic myeloid cell line KBM-7 cells has been exploited to perform large-scale loss-of-function screens in human cells [3,4].

1 citations


Cites background from "Isolation and Characterization of a..."

  • ...Nearhaploid karyotype of chronic myeloid cell line KBM-7 cells has been exploited to perform large-scale loss-of-function screens in human cells [3,4]....

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Posted ContentDOI
16 Nov 2022
TL;DR: Wang et al. as mentioned in this paper developed a mechanism-agnostic method to categorize copy number alterations (CNAs) based on various fragment properties, which reflect the consequences of mutagenic processes and can be extracted from different types of data, including whole genome sequencing (WGS) and SNP array.
Abstract: Abstract Copy number alterations (CNAs) are a predominant source of genetic alterations in human cancer and play an important role in cancer progression. However comprehensive understanding of the mutational processes and signatures of CNA is still lacking. Here we developed a mechanism-agnostic method to categorize CNA based on various fragment properties, which reflect the consequences of mutagenic processes and can be extracted from different types of data, including whole genome sequencing (WGS) and SNP array. The 14 signatures of CNA have been extracted from 2778 pan-cancer analysis of whole genomes (PCAWG) WGS samples, and further validated with 10851 the cancer genome atlas (TCGA) SNP array dataset. Novel patterns of CNA have been revealed through this study. The activities of some CNA signatures consistently predict cancer patients’ prognosis. This study provides a repertoire for understanding the signatures of CNA in cancer, with potential implications for cancer prognosis, evolution, and etiology.

1 citations

Journal ArticleDOI
TL;DR: Current issues in haploid stem cell research are reviewed based on reports published in recent years and the potential applications of these cells in somatic cell nuclear transfer, genome imprinting, and parthenogenesis are assessed.
Abstract: Abstract Haploid embryonic stem cells are embryonic stem cells of a special type. Their nuclei contain one complete set of genetic material, and they are capable of self-renewal and differentiation. The emergence of haploid embryonic stem cells has aided research in functional genomics, genetic imprinting, parthenogenesis, genetic screening, and somatic cell nuclear transfer. This article reviews current issues in haploid stem cell research based on reports published in recent years and assesses the potential applications of these cells in somatic cell nuclear transfer, genome imprinting, and parthenogenesis. Summary Sentence Haploid embryonic stem cells represent a powerful tool for life science research that can be used in genetic screening, functional genomics, somatic cell nuclear transfer, parthenogenesis, and heterozygous cell research.

1 citations

Posted ContentDOI
09 Sep 2021-bioRxiv
TL;DR: In this paper, the authors identify trans-acting modulators of DNA replication timing and demonstrate the remarkable robustness of the human replication timing program and reveal MCM10 as a novel modulator of DNA replication timing.
Abstract: Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. MCM10 mutant cells demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in MCM10-mutant cells was predominantly comprised of replication initiation defects. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel modulator of DNA replication timing.

1 citations

References
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Journal ArticleDOI
16 Jun 1989-Science
TL;DR: The current status of gene targeting with particular emphasis on germ line modification of the mouse genome is discussed, and the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred are described.
Abstract: Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.

2,320 citations

Journal ArticleDOI
28 May 1981-Nature
TL;DR: Clones of homozygous fish have been produced from individual homozygotes and associated genetic methods facilitate genetic analyses of this vertebrate.
Abstract: Homozygous diploid zebra fish have been produced on a large scale by the application of simple physical treatments. Clones of homozygous fish have been produced from individual homozygotes. These clones and associated genetic methods will facilitate genetic analyses of this vertebrate.

1,203 citations

Journal ArticleDOI
08 Aug 1997-Science
TL;DR: At the checkpoint between the prereplicative phase of growth and the phase of chromosome replication, cells lacking p21 failed to arrest the cell cycle in response to DNA damage, but their apoptotic response and genomic stability were unaltered.
Abstract: Most somatic cells die after a finite number of cell divisions, a phenomenon described as senescence. The p21 CIP1/WAF1 gene encodes an inhibitor of cyclin-dependent kinases. Inactivation of p21 by two sequential rounds of targeted homologous recombination was sufficient to bypass senescence in normal diploid human fibroblasts. At the checkpoint between the prereplicative phase of growth and the phase of chromosome replication, cells lacking p21 failed to arrest the cell cycle in response to DNA damage, but their apoptotic response and genomic stability were unaltered. These results establish the feasibility of using gene targeting for genetic studies of normal human cells.

827 citations

Journal Article
TL;DR: The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.
Abstract: Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +1+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-+S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.

473 citations

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