Hprt Locus Research Articles

Stroma-Driven Notch2 Signaling Controls Naïve B Cell Fate By Regulating Microenvironmental Positioning within the Spleen

Marginal zone (MZ) B cells are naïve innate-like B cells that function as a first line of defense in the spleen within the blood-rich marginal zone. Previous research revealed a critical role for Notch2 receptors in B cells and Delta-like1 (Dll1) ligands in Ccl19-Cre + fibroblastic stromal cells to support the development and maintenance of MZ B cells in mice. Yet, little is known about how MZ B cells integrate extracellular signals and Notch-regulated transcriptional programs to support their positioning and function. To understand the basis of Dll1/Notch2's specificity, we used monoclonal antibodies to acutely block Notch2 receptors vs. Dll1 or Dll4 Notch ligands in vivo. Dll1 and Notch2 blockade, but not Dll4 inhibition, rapidly decreased MZ B cell numbers and Notch2-regulated transcription in B cells. Analysis of fluorescent reporter alleles revealed co-expression of Ccl19-Cre;ROSA26 YFP and Dll1-mCherry or Dll4-mCherry within white pulp follicles, showing that both Delta-like ligands are available in fibroblastic stromal cells, although with differential abundance and spatial distribution. To determine if the reliance of MZ B cells on Dll1 was due to ligand availability in the appropriate stromal niche, we inactivated endogenous Dll1 and Dll4 via Ccl19-Cre-mediated recombination, and restored Dll1 or Dll4 expression selectively via separate Cre-inducible expression alleles in the Hprt locus. Dll4 could not sustain Notch2-dependent MZ B cells even when expressed in the correct stromal niche, suggesting that Dll1/Notch2 interactions have unique biochemical and functional properties. As MZ B cells are known to shuttle between the follicle and the MZ across the marginal sinus, we assessed where Dll1/Notch2-mediated signals are delivered to MZ B cells. High abundance of intracellular Notch2 and expression of the Notch target gene, Hes1 (as revealed via a Hes1-GFP reporter) was apparent in CD1d high MZ B cells across the marginal sinus, including prominent signaling within B cell follicles (and not only in the MZ) (Figure 1A). We next evaluated the transcriptional programs controlled by Notch in MZ B cells. Notch2/Dll1-regulated genes included both Myc-dependent and Myc-independent cohorts, with the latter highly enriched for integrin and chemotactic receptor genes including S1pr1, shown previously to guide B cells towards the MZ across the marginal sinus. Myc itself was dispensable for MZ B cell positioning and retention in the spleen. Without S1pr1, B cells experienced Notch signaling within B cell follicles even without entering the MZ. Unlike wild-type B cells, mislocalized S1pr1-deficient MZ-like B cells were retained in the spleen upon Notch deprivation (Figure 1B). These findings identify splenic B cell follicles and not the MZ as a central hub for stroma-driven Dll1/Notch2 signaling, with Notch empowering subsequent B cell migration and positioning to the MZ through a Myc-independent transcriptional program. As many Notch-regulated transcriptional targets are conserved in human Notch-driven B cell lymphomas, we speculate that principles of stroma-driven Notch signaling and its downstream effects in B cells have been conserved during evolution from mouse to human B cells - with the Notch signature tagging the B cell subsets that rely on conserved Notch programs and the B cell malignancies that hijack them.

Abstract 1218: Mercury(II)-induced abnormalities in processing of topoisomerase I-mediated DNA damage

Abstract Mercury (Hg) is a persistent environmental pollutant that has long been associated with neurotoxic and other noncancer health effects in exposed populations. Recent large epidemiological studies using different internal indices of Hg exposure have found significantly elevated risks for skin cancers in both men and women. To understand potential carcinogenic mechanisms for Hg(II), we examined its mutagenicity and impact on processing of different types of DNA damage. In agreement with its lack of DNA reactivity, we found that Hg(II) was not mutagenic at the mammalian Hprt locus and did not change DNA incorporation of rNMPs which are the most abundant endogenous nucleotide abnormalities in the genome. We next tested the impact of Hg(II) at nontoxic or only mildly toxic concentrations on sensitivity of cells to several genotoxicants producing specific classes of DNA damage. We found that even nontoxic concentrations of Hg(II) impaired cellular resistance to topoisomerase I-mediated DNA damage in human epidermal keratinocytes. Hg(II) had no significant effect on cytotoxicity of topoisomerase II-mediated DNA lesions and examination of numerous components of DNA damage response activated by topoisomerase II-targeting drugs showed no major differences between control and Hg(II)-exposed cells. In contrast, Hg(II)-treated keratinocytes displayed a distorted DNA damage response to topoisomerase I-produced DNA damage, manifested in a misbalanced activation of ATM and ATR targets. Consistent with their lower survival, Hg(II)-treated cells accumulated higher levels of toxic DNA double-strand breaks (DSBs). Further analyses indicated that Hg(II) caused impartment of DNA repair of replication-associated DSBs. Thus, although Hg(II) is not directly genotoxic, it can increase genomic instability by interfering with accurate processing of endogenous DNA damage produced by topoisomerase I. Citation Format: Giorgiana Madalina Ursu, Anna M. Cyran, Anatoly Zhitkovich. Mercury(II)-induced abnormalities in processing of topoisomerase I-mediated DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1218.

Microcystin-LR-induced nuclear translocation of cGAS promotes mutagenesis in human hepatocytes by impeding homologous recombination repair

Microcystin-LR (MC-LR) has been recognized as a typical hepatotoxic cyclic peptides produced by cyanobacteria. Nowadays, due to the frequent occurrence of cyanobacterial blooms, the underlying hepatotoxic mechanism of MC-LR has become the focus of attention. In our present work, the mutagenic effect of MC-LR on human normal hepatic (HL-7702) cells regulated by cGAS was mainly studied. Here, we showed that exposure to MC-LR for 1–4 days could activate the cGAS-STING signaling pathway and then trigger immune response in HL-7702 cells. Notably, relative to the treatment with 1 μM MC-LR for 1–3 days, it was observed that when HL-7702 cells were exposed to 1 μM MC-LR for 4 days, the mutation frequency at the Hprt locus was remarkably increased. In addition, cGAS in HL-7702 cells was also found to complete the nuclear translocation after 4-day exposure. Moreover, co-immunoprecipitation and homologous recombination (HR)-directed DSB repair assay were applied to show that homologous recombination repair was inhibited after 4-day exposure. However, the intervention of the nuclear translocation of cGAS by transfecting BLK overexpression plasmid restored homologous recombination repair and reduced the mutation frequency at the Hprt locus in HL-7702 cells exposed to MC-LR. Our study unveiled the distinct roles of cGAS in the cytoplasm and nucleus of human hepatocytes as well as potential mutagenic mechanism under the early and late stage of exposure to MC-LR, and provided a novel insight into the prevention and control measures about the hazards of cGAS-targeted MC-LR.

Comparative evaluation of three methylene dianiline isomers in the bacterial reverse mutation assay, the in vitro gene mutation test, and the in vitro chromosomal aberration test.

4,4'-MDA is classified as a genotoxic carcinogen based on numerous in vitro and animal data. The consequential assumption that a safe threshold does not exist is not only applied to 4,4'-MDA but also to its structural isomers and impurities 2,2'- and 2,4'-MDA in the absence of substance-specific data. This constitutes a problem in human risk assessments for all three substances as the inherent risks of 2,2'- and 2,4'-MDA and their contribution as impurities to that of 4,4'-MDA are essentially unknown. A comparative in vitro genotoxicity dataset consisting of the bacterial reverse mutation (Ames) test and the chromosomal aberration test in human lymphocytes (both performed according to the current OECD Guidelines) was generated for all three isomers. Furthermore, an in vitro gene mutation test in Chinese hamster ovary (CHO) cells (HPRT locus assay) was conducted with 2,4'-MDA. The results indicate differences regarding the genotoxic mechanism and potential, respectively, between the three structures and suggest that the no-threshold assumption for 4,4'-MDA may not be appropriate for 2,2'- and 2,4'-MDA.

Cytotoxicity and genotoxicity of blue LED light and protective effects of AA2G in mammalian cells and associated DNA repair deficient cell lines

Light emitting diode (LED) devices emit narrow bands of the blue, green, and red light spectrum rather than the continuous spectrum emitted from sunlight and fluorescent light bulbs. LED devices have become considerably common in society, and the fluence of blue light from LED devices is more intense than other light sources. Previous studies presented that the blue light spectrum may harness potentially inimical genotoxicity. Therefore, the aim of this study was to investigate this potential cytotoxicity and genotoxicity, as well as identify the mechanism of the cellular effects induced by blue LED light exposure in mammalian cell lines with their DNA repair deficient mutants. Our results demonstrated that blue LED light induced both oxidative stress to cells and cytotoxic and genotoxic effects including reduction of clonogenicity, cell cycle arrest, induction of sister chromatid exchanges, endoreduplicated chromosomes, and increased frequency of HPRT locus mutations. In DNA repair deficient cells, particularly those involving double strand break repair deficiency, cells presented hypersensitivity to blue LED light exposure. Blue LED light also induced chromosome aberrations more in DNA repair deficient cells than wild type cells. The cytotoxicity of blue LED light was reduced by an effective antioxidant, ascorbic acid 2-glucoside, which can suppress blue LED light induced oxidative stress. These results indicated that prolonged, high intensity exposure to blue LED light induces genotoxic stress to cells, and oxidative stress induced by blue LED light is targeting DNA to induce these biological effects.

Visualization of X chromosome reactivation in mouse primordial germ cells in vivo.

ABSTRACTX chromosome inactivation (XCI), determined during development, remains stable after embryonic cell divisions. However, primordial germ cells (PGCs) are exceptions in that XCI is reprogrammed and inactivated X chromosomes are reactivated. Although interactions between PGCs and somatic cells are thought to be important for PGC development, little is known about them. Here, we performed imaging of X chromosome reactivation (XCR) using the ‘Momiji’ mouse system, which can monitor the X chromosome's inactive and active states using two color fluorescence reporter genes, and investigated whether interactions would affect XCR in PGCs. Based on their expression levels, we found that XCR of the Pgk1 locus began at embryonic day (E)10.5 and was almost complete by E13.5. During this period, PGCs became distributed uniformly in the genital ridge, proliferated, and formed clusters; XCR progressed accordingly. In addition, XCR of the Pgk1 locus preceded that of the Hprt locus, indicating that the timing of epigenetic memory erasure varied according to the locus of each of these X-linked genes. Our results indicate that XCR proceeds along with the proliferation of PGCs clustered within the genital ridge.This article has an associated First Person interview with the first author of the paper.

Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

The Gata1low murine megakaryocyte–erythroid progenitor cells expand robustly and alter differentiation potential

GATA1 is a master transcription factor of megakaryopoiesis and erythropoiesis, and loss-of-function mutation can induce accumulation of megakaryocyte–erythroid progenitors (MEPs) in mice and humans. Accordingly, the murine MEP cell line (termed G1ME2 cells) encoding doxycycline (dox)-inducible anti-Gata1 shRNA on Hprt locus has been developed. The cells were CD41+CD71+KIT+, expand under dox, stem cell factor, and thrombopoietin (TPO), and terminally differentiate into erythroid cells or megakaryocytes upon removal of dox. Surprisingly, in this study, these Gata1low murine MEPs displayed accelerated growth from around 90–100 days after cell culture, impeded megakaryocytic potential, and maintained erythropoiesis. We specified them as late G1ME2 cells and discovered that increased CD41−KIT+ population during long-term culture was the main reason for the delayed megakaryopoiesis. The CD41 expression level was partially de-repressed by PI3K/AKT inhibitors, suggesting that TPO-mediated cell survival signaling pathway might have impacted on CD41 in the late G1ME2 cells. Nevertheless, among the late cells, the CD41+KIT+ cells could still generate megakaryocytes on dox withdrawal. Taken together, G1ME2 cells could provide a good model to study molecular mechanism of hematopoiesis because of their ability to expand excessively without artificial immortalization.

Gene (HPRT) and chromosomal (MN) mutations of nickel metal powder in V79 Chinese hamster cells

Nickel metal is a naturally occurring element used in many industrial and consumer applications. Human epidemiological data and animal cancer bioassays indicate that nickel metal is not likely to be a human carcinogen. Yet, nickel metal is classified as a suspected human carcinogen (CLP) and possibly carcinogenic to humans (IARC). There are no reliable studies on the potential for nickel metal to induce gene and micronucleus (MN) mutations. To fill these datagaps and increase our understanding of the mechanisms underlying the lack of nickel metal carcinogenicity, gene and micronucleus mutation studies were conducted with nickel metal powder (N36F) in V79 Chinese Hamster cells following OECD 476 and 487 guidelines, respectively, under GLP. Gene mutation at the hprt locus was tested, with and without metabolic activation, after 4-h treatment with 0.05–2.5 mM nickel metal powder. Cytokinesis-block MN frequency following exposure to 0.25–1.5 mM nickel metal was tested after 4-h treatment, with and without metabolic activation, followed by a 24-h treatment without metabolic activation. In the gene mutation assay, there were modest increases in hprt mutants observed at some test concentrations, not exceeding 2.2-fold, which were either within the historical control values and/or showed no concentration-response trend. The positive controls showed increases of at least 7-fold. Likewise, no increases in the MN frequency exceeding 1.5-fold were observed with nickel metal, with no concentration-response trends. Taking these results together, it can be concluded that nickel metal is non-mutagenic and does not cause gene nor chromosomal mutations.

A critical Assessment of the Genotoxicity Profile of the Fungicide Tricyclazole.

Tricyclazole (8-methyl-[1,2,4]triazolo[3,4-b][1,3]benzothiazole) is a fungicide used globally on rice for treatment of the seasonal rice blast disease. Human exposure to this fungicide can occur via dietary and nondietary routes. In a battery of in vitro assays, tricyclazole did not induce gene mutations in bacteria (Ames test) or at the Hprt locus of CHO cells. It was also negative for the induction of micronuclei in human lymphocyte cultures and unscheduled DNA synthesis (UDS) in primary rat hepatocyte. Paradoxically, tricyclazole induced a mutagenic response at the Tk locus of the mouse lymphoma L5178Ycells (MLA), which occurred equally among small/large colony phenotypes. Selection of preexisting mutants leading to a false-positive response in the MLA was ruled out in follow-up experiments. In vivo, tricyclazole was negative in the rat liver UDS assay, mouse bone micronucleus test and a transgenic (MutaMouse) gene mutation assay in glandular stomach, liver, and kidney. Other supporting evidence for the lack of genotoxicity for tricyclazole comes from an in vivo study for sister chromatid exchanges in Chinese hamsters, and a dominant lethal test in the male germ cells of mice. The combined evidence from the genotoxicity studies together with the evidence from toxicokinetic, carcinogenicity, developmental, and reproductive toxicity studies confirm that mutagenicity does not occur in relevant in vivo systems. Data were also compared to potential animal and human exposure, mechanistic data on biological targets and data on analogues, confirming adequacy of the available data for hazard identification and risk assessment. Environ. Mol. Mutagen. 61:300-315, 2020. © 2019 Wiley Periodicals, Inc.

Role of Human N-Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen-Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay.

Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc.

The IWGT in vitro Mammalian Cell Gene Mutation (MCGM) assays working group—Introductory remarks & consensus statements

Assays for gene mutations in cultured mammalian cells, i.e., Mammalian Cell Gene Mutation (MCGM) assays, are widely used in genetic toxicology laboratories worldwide; over the past four decades they have been commonly employed in safety assessment studies, and studies designed to address hypothesis-driven research questions. Despite many advances in the fields of cellular and molecular biology over the past four decades, the MCGM assays commonly used for regulatory evaluations continue to be those developed in the 1970s, including assays that enumerate induced mutations at the hprt or tk loci of several commonly used cell lines. Consequently, the Steering Committee of the 7th International Workshops on Genotoxicity Testing (IWGT) convened a working group (WG) to critically assess the state-of-the-science in regards to the current and emerging tools for the detection of mutagens using cultured mammalian cells. The WG was divided into four sub-groups that evaluated the state-of- the-science with respect to the: (1) in vitro Pig-a gene mutation assay, (2) in vitro assays based on cells from transgenic rodents, (3) technologies and innovations to improve MCGM assays using TK6 cells, and (4) novel and emerging technologies and approaches for detection and enumeration of gene mutations in mammalian cells. Each of these sub-groups critically reviewed the scientific literature, along with other unpublished data, to develop consensus statements on the status of the test systems in their respective focus areas. These reviews, with their associated consensus statements, are presented in the accompanying works by Bemis and Heflich., White et al., Honma et al., and Evans et al. The MCGM assay WG, in consultation with the entire IWGT, formulated consensus statements regarding the overall utility of MCGM assays for identification and assessment of mutagenic hazard.

756 ATR kinase activity promotes the viability and proliferative capacity of quiescent human keratinocytes exposed to UVB radiation

The ATR protein kinase plays well-described roles during the DNA synthesis phase of the cell cycle in maintaining genomic integrity in response to DNA damage induced by UV radiation and other genotoxic agents. However, the function and activation mechanism of ATR in cells that are not actively replicating DNA remains largely unexplored but is physiologically important and relevant to understanding the fate of quiescent and stem cell populations exposed to DNA damaging agents. Using HaCaT and telomerase-immortalized normal human keratinocytes maintained in a confluent, non-replicating state in vitro, we show that the ATR kinase is rapidly activated in response to UVB radiation in a manner dependent on the DNA translocase XPB. Inhibition of ATR kinase activity has modest effects on acute cell survival but more severely abrogates the ability of UVB-irradiated keratinocytes to proliferate upon stimulation with growth factors. Furthermore, ATR kinase inhibition in quiescent HaCaT keratinocytes potentiates UVB mutagenesis at the HPRT locus but does not impact the rate of removal of cyclobutane pyrimidine dimers from genomic DNA. These results indicate that the ATR kinase plays important roles in limiting mutagenesis and promoting cell viability and proliferation following UVB exposure even in cells that are not actively synthesizing DNA.

In Vitro Approaches for Assessing the Genotoxicity of Nanomaterials.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Abstract 5111: Gene editing in the NSG mouse strain and its genetic derivatives

Abstract Mouse models continue to be powerful tools in preclinical oncology and immuno-oncology studies. The development of the inbred highly immunocompromised mouse strain NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) has dramatically improved the ability to work with human tumors in an organismal context using xenograft approaches. Furthermore, technical innovations have expanded the utility of this personalized tumor platform via the reconstitution of parts of the human immune system. An example of this is the NSG-SGM3 inbred mouse strain, in which transgenes expressing human IL3, GMCSF and SCF have been stably integrated into the NSG background. This NSG “derivative” strain can effectively support the stable engraftment of human myeloid lineages and the regulatory T cell populations. Direct genetic modification of the NSG genome in mouse embryos was a key technical capability to attain this NSG “derivative” strain rapidly and avoid extensive breeding. Modification of NSG and derivative strains represent an opportunity to optimize the engraftment of other human lineages. The ability to apply modern genome modification technology to different inbred mouse strains is highly desirable but must overcome two major technical hurdles - 1) Reproductive biology constraints that prevent live born progeny after reagent delivery methods such as microinjection or electroporation and 2) Cell intrinsic DNA-repair processes that are a prerequisite enabling gene editing tools. Given the SCID mutation (Prkdcscid) in the NSG background compromises some DNA repair pathways, it was not clear whether nuclease assisted recombination technologies would be a robust tool to modify NSG (and derivatives) genomes at the zygote stage. Here, we demonstrate that CRISPR-Cas9 is an effective tool for introducing genome modifications in NSG and present a specific example using the Hprt locus. Furthermore, we will present general experience with genomic modifications using NSG, and its derivatives, to emphasize direct modification of these genomes is technically feasible and is enabling the rapid sequential genetic tailoring in this high value cancer model strain. Citation Format: David S. Grass, F William Buaas, Michael V. Wiles, Benjamin E. Low, Peter Kutny. Gene editing in the NSG mouse strain and its genetic derivatives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5111.

Cre-Mediated Transgene Integration in Chinese Hamster Ovary Cells Using Minicircle DNA Vectors.

Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2-3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells.

Gene Editing in the NSG mouse strain and its genetic derivatives

Abstract The NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mouse strain is highly immunodeficient and has dramatically improved the ability to work with human tumors in an organismal context using xenograft approaches. Technical innovations have expanded the utility of this personalized tumor platform via the reconstitution of parts of the human immune system. An example of this is the NSG-SGM3 inbred mouse strain, in which transgenes expressing human IL3, GMCSF and SCF have been stably integrated into the NSG background, and can effectively support the stable engraftment of human myeloid lineages and the regulatory T cell populations. Direct modification of the NSG genome in mouse embryos was a key to attain this NSG “derivative” strain rapidly and avoid extensive breeding. Modification of NSG, which is built on the parental NOD/ShiLtJ background, a commonly utilized polygenic model of autoimmune type 1 diabetes, can optimize the engraftment of other human lineages. Modern genome modification technology must overcome strain specific characteristics. DNA repair is involved in creating mutations by resolving double strand breaks. The SCID mutation (Prkdcscid) in the NSG background compromises some DNA repair pathways. It was not clear whether nuclease assisted recombination technologies would be a robust tool to modify NSG genomes at the zygote stage. We demonstrate CRISPR-Cas9 is an effective tool for introducing genome modifications in NSG and present a specific example using the Hprt locus. We will also present our experience modifying NSG and its derivatives, demonstrating that direct modification of these genomes is technically feasible and is enabling the rapid sequential genetic tailoring in this high value immunodeficient strain.

Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.

A novel Cre-inducible knock-in ARL13B-tRFP fusion cilium reporter.

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre-inducible cilium-specific reporter mouse line expressing an ARL13B-tRFP fusion protein driven by a CMV enhancer/chicken β actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono- and multiciliated tissues and by time-lapse live-cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live-cell imaging. Thus, the ARL13B-tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue-specific manner to understand processes, such as ciliary protein trafficking or cilium-dependent signaling in vitro and in vivo.

Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells

We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production.