CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells

Xia, Baolong; Amador, Gabriel; Viswanatha, Raghuvir; Zirin, Jonathan; Mohr, Stephanie E.; Perrimon, Norbert

doi:10.1038/s41596-020-0383-8

Cited by 6 publications

(4 citation statements)

References 28 publications

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“…We considered using a Crispr-Cas9 approach in the hybridoma cell line for a clean knockout of FKBP11, which ultimately may have led to more conclusive results on the role of FKBP11 in antibody synthesis and secretion. But as CrisprCas9 approaches are a challenging task in polyploid cells, we ultimately decided against that, given the polyploid nature of the hybridoma cell line [82]. As to in vivo models, the FKBP11 knockout mouse is, to the best of our knowledge, not yet commercially available.…”

Section: Discussionmentioning

confidence: 99%

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis

Preisendörfer

Ishikawa

Hennen

et al. 2022

Cells

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Antibodies are central effectors of the adaptive immune response, widespread used therapeutics, but also potentially disease-causing biomolecules. Antibody folding catalysts in the plasma cell are incompletely defined. Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease with increasingly recognized autoimmune features. We found elevated expression of FK506-binding protein 11 (FKBP11) in IPF lungs where FKBP11 specifically localized to antibody-producing plasma cells. Suggesting a general role in plasma cells, plasma cell-specific FKBP11 expression was equally observed in lymphatic tissues, and in vitro B cell to plasma cell differentiation was accompanied by induction of FKBP11 expression. Recombinant human FKBP11 was able to refold IgG antibody in vitro and inhibited by FK506, strongly supporting a function as antibody peptidyl-prolyl cis-trans isomerase. Induction of ER stress in cell lines demonstrated induction of FKBP11 in the context of the unfolded protein response in an X-box-binding protein 1 (XBP1)-dependent manner. While deficiency of FKBP11 increased susceptibility to ER stress-mediated cell death in an alveolar epithelial cell line, FKBP11 knockdown in an antibody-producing hybridoma cell line neither induced cell death nor decreased expression or secretion of IgG antibody. Similarly, antibody secretion by the same hybridoma cell line was not affected by knockdown of the established antibody peptidyl-prolyl isomerase cyclophilin B. The results are consistent with FKBP11 as a novel XBP1-regulated antibody peptidyl-prolyl cis-trans isomerase and indicate significant redundancy in the ER-resident folding machinery of antibody-producing hybridoma cells.

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Section: Discussionmentioning

confidence: 99%

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis

Preisendörfer

Ishikawa

Hennen

et al. 2022

Cells

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“…A pair of targeting sites of small guide RNAs (sgR-NAs) located in exon 2 of gpr56 were designed, namely, Target 1 and Target 2 (Figure 1A). They were designed with the expectation that the CRISPR/Cas9 gene-editing system was used to introduce frameshift mutations into the coding sequence (CDS) of gpr56 through nonhomologous end joining (NHEJ), resulting in translation errors and premature stop codons of gpr56 [21,22]. A transcription template of the sgRNAs targeting exon2 of gpr56 was synthesized via PCR (the primers used are shown in Table S1).…”

Section: Construction Of Gpr56-knockout Zebrafish Linesmentioning

confidence: 99%

Transcriptomics and Phenotypic Analysis of gpr56 Knockout in Zebrafish

Sun

Yang

Zheng

et al. 2023

IJMS

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The adhesion G-protein-coupled receptor is a seven-transmembrane receptor protein with a complex structure. Impaired GPR56 has been found to cause developmental damage to the human brain, resulting in intellectual disability and motor dysfunction. To date, studies on gpr56 deficiency in zebrafish have been limited to the nervous system, and there have been no reports of its systemic effects on juvenile fish at developmental stages. In order to explore the function of gpr56 in zebrafish, the CRISPR/Cas9 gene-editing system was used to construct a gpr56-knockout zebrafish. Subsequently, the differentially expressed genes (DEGs) at the transcriptional level between the 3 days post fertilization (dpf) homozygotes of the gpr56 mutation and the wildtype zebrafish were analyzed via RNA-seq. The results of the clustering analysis, quantitative PCR (qPCR), and in situ hybridization demonstrated that the expression of innate immunity-related genes in the mutant was disordered, and multiple genes encoding digestive enzymes of the pancreatic exocrine glands were significantly downregulated in the mutant. Motor ability tests demonstrated that the gpr56−/− zebrafish were more active, and this change was more pronounced in the presence of cold and additional stimuli. In conclusion, our results revealed the effect of gpr56 deletion on the gene expression of juvenile zebrafish and found that the gpr56 mutant was extremely active, providing an important clue for studying the mechanism of gpr56 in the development of juvenile zebrafish.

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“…reflects the challenges involved in the genome manipulation of these parasites [114,115]. However, the recent advent of genome editing using CRISPR-Cas9 and its variations should help resolve some of these functional questions that are still lingering, as has been done for other genes and in other species [116][117][118]. In addition, the available evidence on the subject matter shows some differences in the TERT between members of the trypanosomatid family and may be due to the enzyme s usefulness to the parasite [89,119].…”

Section: Phylogenetic Context Of Leishmania Spp Telomerases Among Other Pathogenic Trypanosomatidsmentioning

confidence: 99%

Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?

Assis

Andrade-Silva

Shiburah

et al. 2021

Cells

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Leishmaniases belong to the inglorious group of neglected tropical diseases, presenting different degrees of manifestations severity. It is caused by the transmission of more than 20 species of parasites of the Leishmania genus. Nevertheless, the disease remains on the priority list for developing new treatments, since it affects millions in a vast geographical area, especially low-income people. Molecular biology studies are pioneers in parasitic research with the aim of discovering potential targets for drug development. Among them are the telomeres, DNA–protein structures that play an important role in the long term in cell cycle/survival. Telomeres are the physical ends of eukaryotic chromosomes. Due to their multiple interactions with different proteins that confer a likewise complex dynamic, they have emerged as objects of interest in many medical studies, including studies on leishmaniases. This review aims to gather information and elucidate what we know about the phenomena behind Leishmania spp. telomere maintenance and how it impacts the parasite’s cell cycle.

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CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells

Cited by 6 publications

References 28 publications

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis

Transcriptomics and Phenotypic Analysis of gpr56 Knockout in Zebrafish

Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?

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