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First molecular cytogenetic high resolution characterization of the NIH 3T3 cell line by murine multicolor banding.

ABSTRACT: Since being established in 1963, the murine fibroblast cell line NIH 3T3 has been used in thousands of studies. NIH 3T3 immortalized spontaneously and became tetraploid shortly after its establishment. Here we report the first molecular cytogenetic characterization of NIH 3T3 using fluorescence in situ hybridization based multicolor banding (mcb). Overall, a complex rearranged karyotype presenting 16 breakpoints was characterized. Also it was possible to deduce the resulting gains and losses of copy numbers in NIH 3T3. Overall, only 1.8% of the NIH 3T3 genome is disome, 26.2% tri-, 60% tetra-, 10.8% quinta-, and 1.2% hexasome. Strikingly, the cell line gained only 4 derivative chromosomes since its first cytogenetic description in 1989. An attempt to align the observed imbalances of the studied cell line with their homologous regions in humans gave the following surprising result: NIH 3T3 shows imbalances as typically seen in human solid cancers of ectodermal origin.

SUBMITTER: Leibiger C

PROVIDER: S-EPMC3621507 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line.

Project description:BackgroundCD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study.MethodsTotal RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic.ResultsAmplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively).ConclusionStable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera.

| S-EPMC4388889 | biostudies-literature

NIH-3T3 cell line response to proteasome inhibitor

Project description:In this study, we compared the transcriptional profile of NIH-3T3 cells that are either wild-type (WT) or Nrf1 knockout (Nrf1-KO). We treated these cells with carfilzomib for 6 hours or 24 hours and performed RNA-sequencing with these samples.

2020-05-21 | GSE144817 | GEO

Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis.

Project description:The information that is obtained from single cells during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis is influenced by the method that was used to prepare the cells. The removal of extracellular media before analysis is necessary, but the rinsing technique should not damage the plasma membrane of the cell. The presence of intracellular salts reduced the secondary ion yield an average of 2.6-fold during Bi3 (+)/C60 (++) depth profiles. Chemical fixation followed by rinsing removed a majority of the intracellular salts, "recovering" the positive secondary ion yields. The formaldehyde-fixation process removed a majority of the intracellular Cl(-), but other key anions were not removed in significant amounts. The data presented here is consistent the anion neutralization mechanism largely responsible for the lower ion yields. All of the organic secondary ions that were detected in the freeze-dried cells were also detected in the formaldehyde-fixed cells, suggesting that the fixation process did not remove any molecular species to an extent that is detectable by ToF-SIMS. Compared to freeze dried cells, well preserved, frozen-hydrated cells showed little increase, or a decreased yield, for most low mass ions, but an increased yield for larger mass fragments. This is consistent with a reduced damage cross section at cryogenic analysis temperatures, although proton donation from water and reduction the salt effects in the presence of water likely also play roles. Numerous ions detected from the frozen-hydrated cells were not detected from the freeze dried cells, however many of these ions were attributed to chemical combinations of water, salts and the ammonium acetate rinsing solution.

| S-EPMC4000548 | biostudies-literature

Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts.

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| S-EPMC5302943 | biostudies-literature

Generation of multicolor banding probes for chromosomes of different species.

Project description:UnlabelledBackgroundThe multicolor banding (MCB/mBAND) technique provides a unique opportunity to characterize intrachromosomal rearrangements and to determine chromosomal breakpoints. Until recently, MCB probes have only been available for human and some murine chromosomes. Generation of MCB probes for chromosomes of other species, useful and required in many cytogenetics research fields, was limited by technical difficulties. MCB probes are established by chromosome microdissection followed by whole genomic DNA amplification. However, unambiguous identification of the target chromosome is required for MCB-probe establishment. Previously proposed protocols suggested G-banding staining or preliminary FISH with whole chromosome paints (WCP) as methods to identify the chromosome of interest.ResultsHere we present a complete workflow for MCB probe generation for those cases and species where chromosome morphology is too challenging to recognize target chromosomes by conventional methods and where WCP probes are not available. The workflow was successfully applied for murine chromosomes that are difficult to identify unambiguously. Additionally, we showed that glass-needle based microdissection enables establishment of a whole set of WCP paints by microdissection of individual chromosomes of a single metaphaseConclusionsThe present method can be applied for generation of whole or region-specific DNA probes for species, where karyotyping of G-banded chromosomes is challenging due to similar chromosome morphology and/or chromosome banding patterns.

| S-EPMC3575270 | biostudies-literature

RNA-Seq of murine NIH/3T3 cells upon Aldoa knockdown

Project description:Aldoa knockdown (using a pool of 4 siRNAs) vs. non-targeting control knockdown (using a pool of 4 siRNAs) in murine NIH/3T3 cells (fibroblasts). Cells were harvested 48 hours after transfection.

2022-07-13 | GSE207899 | GEO

NIH 3T3: Ctrl vs Reo

Project description:Transcriptional profiling of NIH 3T3 comparing WT, RIG-I KD with and without reovirus infection

2020-10-07 | GSE127850 | GEO

NIH-3T3 cell line response to proteasome inhibitor

Project description:NIH-3T3 cell line response to proteasome inhibitor

| PRJNA604998 | ENA

Molecular cytogenetic characterization of the urethane-induced murine lung cell line LA-4 as a model for human squamous cell lung cancer.

Project description:The murine tumor cell line LA-4 (also known as LA4 or LA 4) has been extensively used in ~70 studies from 1975 to present. However, the genetic characteristics have not been comprehensively delineated, apart from a single solid-stain cytogenetic study, which reported an average of 116 chromosomes per cell. LA-4 was created via urethane induction in an A/He mouse and demonstrated characteristic features of lung adenoma cells. In the present study, multicolor banding-based molecular cytogenetics was combined with molecular karyotyping to characterize ploidy, copy number alterations and chromosomal breakpoints of LA-4. A hyper-tetraploid karyotype with 85-93 chromosomes per cell, three distinct (pseudo-) dicentric derivatives, two neocentrics, four unbalanced translocations, two chromosomes with terminal deletions and one chromosome with a balanced inversion were detected. The results were translated into the human genome and a comparison with the literature revealed that LA-4 is well-suited as a murine model for human squamous cell lung cancer.

| S-EPMC8647198 | biostudies-literature

Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv).

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| S-EPMC3966832 | biostudies-literature

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