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Results for “Clone tracing”

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At least 19 recordsLinked to original sources

PubMed2025

SpaceBar enables clone tracing in spatial transcriptomic data.

We report a cellular barcoding strategy, SpaceBar, that enables simultaneous clone tracing and spatial transcriptomics profiling. Our approach uses a library of 96 synthetic barcode sequences that can be robustly detected by imaging based spatial transcriptomics (seqFISH), delivered such that each cell is labeled with a combination of barcodes. We used these barcodes to label melanoma cells in a tumor xenograft model and profiled both clone identity and spatial gene expression in situ. We developed a gene scoring metric that quantifies how strongly gene expression is driven by intrinsic cellular cues or extrinsic environmental signals. Our framework distinguishes between clonal dynamics and environmentally-driven transcriptional regulation in complex tissue contexts.

Journal Article↗

PubMed2025

Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.

ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.

Animals↗

PubMed1978

Distribution of immunoglobulin-containing cells in bone marrow and lymphoid tissues in patients with monoclonal gammapathy.

Cell suspensions of bone marrow and lymphoid tissue from 85 patients with monoclonal gammapathy were investigated by a direct immunofluorescence procedure for the detection of intracellular immunoglobulin alpha, mu, gamma, kappa and lambda chains. Serum Ig levels were determined and daily syntheric rates estimated. In all cases the majority of Ig-containing bone marrow cells contained the same Ig class as that of the M-component in serum or urine indicating a diffuse distriubtion of these clones in the bone marrow. This was observed not only in myeloma but also in benign monoclonal gammapathy (BMG) and lymphoma with an M-component. The M-component producing clone could be traced to extramedullary lymphoid tissue in myeloma but usually not in BMG. A positive correlation was found between the calculated synthetic rate of the M-component and the number of Ig-containing cells in the bone marrow and some indication was found that the synthetic rate per cell might be lower in IgM and IgG than in IgA monoclonal gammapathy. The depressed level of polyclonal Ig in myeloma and to some extent in BMG was parallelled by a diminished number of Ig-containing cells in the bone marrow.

Adult↗

PubMed1975

In vitro growth of neoplastic rat glial cells.

An ependymoma was induced in the brain of a BD-IX rat by repeated doses of MNU. In vitro grown cells of the tumor were fibroblasts and stellate cells considered to be glial. Reimplantation of these cells into the brain of another BD-IX rat resulted a tumor growing in the meninges. This tumor was then repeatedly transplanted over several passages. Its gross morphology resembled that of a sarcoma; however, glial in cells could be demonstrated with silver methods. A cell line derived from the first transplanted tumor group proved to contain S-100 protein in traces in the first passages in vitro. Explanted cells of the primary ependymoma were cloned. Two clones were established, RGL 1 and RGL 2. The cells of both clones resembled morphologically the stellate elements of the primary explant. RGL 1 contained S-100 protein. It was cloned again in the 16th subpassage. Four clones were established. Only 1 contained S-100 protein in traces. The clone RGL 1 had a subdiploid karyotype with a modal number of 39 and a large submetacentric marker. It was subcloned in the 25th passage with two clones, both having a karyotype of 41 chromosomes.

Animals↗

PubMed2025

Cloning and validating systems for high throughput molecular recording.

Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.

Cloning, Molecular↗

PubMedDate not supplied

Age-dependent micronuclear deterioration in Tetrahymena pyriformis, syngen 1.

In the ciliate Tetrahymena pyriformis, syngen 1, senescence is marked both by mitotic and meiotic cytogenetic irregularities of the micronucleus (germinal nucleus) and by an increased frequency of cell death. Full vigor can be restored through the age-dependent process of genomic exclusion, which in prematurely senscent clones C, A III and A V results in the replacement of defective micronuclei but not in the development of new macronuclei (somatic nuclei). After a period of normal behavior, which lasts about 40 fissions in C and 100 fissions in A III and A V, the new micronucleus placed in the same cytoplasm as the old macronucleus again becomes defective and the rejevenated clone again shows signs of senescence. The origin of these defective micronuclei is traced to action of the old macronucleus. It is suggested that the time of course of aging is regulated through the accumulation of unrepaired damage to the micronuclear genetic material.

Aging↗

PubMed1977

Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein.

A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 micrograms per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium.

Adenine↗

PubMed2026

Aging and DNA damage are associated with the development of endothelial cell clonal expansion.

Endothelial dysfunction is a hallmark of vascular aging and a key contributor to cardiovascular disease. Although senescence has been widely studied as a terminal endothelial cell fate, recent evidence suggests that clonal expansion, the proliferative expansion of genetically identical cells, may also occur in aged tissues. We sought to determine whether endothelial clonal expansion increases with age, specifically at the atheroprone regions of the aorta, and to evaluate whether DNA damage promotes endothelial cell clonal expansion. Tamoxifen-inducible, endothelial-specific Cdh5-CreERT2 male and female mice were used to quantify clonal expansion in endothelial cells (ECs) across the aortic region in both young (4 mo) and aged (24 mo) mice. We further examined the effect of DNA damage by administering systemic doxorubicin (DOXO) to assess clonal dynamics in different aortic regions. Aging significantly increased EC clone size and the percentage of clonal ECs in atheroprone regions, particularly the minor arch, whereas only clone size increased in nonatheroprone regions. Systemic DOXO administration increased clone size across the aortic region without altering clonal recruitment, indicating selective amplification of preexisting clones. These findings suggest that clonal expansion is promoted by both aging and DNA damage. Clonal expansion may represent an underrecognized mechanism contributing to endothelial homogeneity and vascular remodeling during aging and in response to sublethal genomic stress.NEW & NOTEWORTHY Aging reshapes the vascular endothelium in unexpected ways. Using lineage tracing in mice, we show that endothelial cells undergo age-dependent clonal expansion, particularly in atheroprone regions exposed to disturbed blood flow. This process is amplified by DNA damage and reflects the selective expansion of preexisting clones rather than increased recruitment. Endothelial clonal expansion may represent an underrecognized mechanism driving vascular remodeling during aging and genotoxic stress.

Animals↗

PubMed2025

Sustained NF-κB activation allows mutant alveolar stem cells to co-opt a regeneration program for tumor initiation.

Disruptions to regulatory signals governing stem cell fate open the pathway to tumorigenesis. To determine how these programs become destabilized, we fate-map thousands of murine wild-type and KrasG12D-mutant alveolar type II (AT2) stem cells in vivo and find evidence for two independent AT2 subpopulations marked by distinct tumorigenic capacities. By combining clonal analyses with single-cell transcriptomics, we unveil striking parallels between lung regeneration and tumorigenesis that implicate Il1r1 as a common activator of AT2 reprogramming. We show that tumor evolution proceeds through the acquisition of lineage infidelity and reversible transitions between mutant states, which, in turn, modulate wild-type AT2 dynamics. Finally, we discover how sustained nuclear factor κB (NF-κB) activation sets tumorigenesis apart from regeneration, allowing mutant cells to subvert differentiation in favor of tumor growth.

Animals↗

PubMed1978

The localization of mouse globin genes: a test of the effectiveness of hybridization in situ.

Hybridization of rabbit reticulocyte mRNA to banded mouse chromosomes in situ labeled several regions, including the globin loci. Whereas the labeling was sufficient to detect unknowns in the globin size class, the chromosome assignments would be doubtful without some means of removing trace contaminants from the probe or of recognizing chromosomal regions to which they hybridize. Mammalian gene mapping by means of hybridization in situ might be feasible with probes cloned in microbial host-vector systems or with kinetic analysis of the hybridization process at every labeled site.

Animals↗

PubMed2025

Combined somatic mutation and transcriptome analysis reveals region-specific differences in clonal architecture in human cortex.

The human cerebral cortex is specialized into regions, but little is known about how human cellular lineages shape cortical regional variation and neuronal cell-type distribution during development. Here, we map single-cell lineages of human cortical regions and neuronal subtypes using >1,000 somatic single-nucleotide variants (sSNVs) identified from deep bulk whole-genome sequencing and analyzed over 25 regions and >72,000 single cells. In the fronto-parietal cortex, sSNVs are rarely restricted, marking neuron-generating clones that disperse into neighboring regions. In contrast, the primary visual cortex harbors 30%-70% more sSNVs than the neighboring secondary visual cortex. Clones at this border exhibit more restricted dispersion, suggesting late developmental lineage segregation. Single-nucleus sSNV and whole-transcriptome analysis reveal glutamatergic neuron clones with modest regional restrictions that share low-mosaic sSNVs with some GABAergic neurons, suggesting a recent dorsal cortical progenitor. Our analysis reveals human-specific cortical lineage patterns, regional differences in clonal patterns, and late divergence of some glutamatergic/GABAergic lineages.

Humans↗

PubMed1978

The morphology and kinetics of spermatogonial degeneration in normal adult rats: an analysis using a simplified classification of the germinal epithelium.

The phenomena of spermatogonial degeneration have been studied in normal adult rat testes using a simplified classification of the germinal epthelium based upon the six types of differentiating spermatogonia. The following features distinguished this from schemes based on acrosome development. Rather than 14 states of unequal duration, there are only six stages, five of which are the same length. The classification starts at the beginning of spermatogenesis with A1 spermatogonia rather than at the onset of spermiogenesis. The classification is derived from acutal biological events in spermatogenesis, namely generation times of spermatogonia, rather than upon arbitrary events in acrosome development. Most importantly, this new classification can be used with most types of preparations and in most experimental conditions. Examination of tubular whole mounts reveals that degeneration preferentially occurs in types A2 and A3 and to a lesser extent A4 spermatogonia, and is rarely seen in generations of A1, In or B cells. Deterioration is first manifested in clusters of cells joined by the intercellular bridges as they complete DNA synthesis and enter the 2 phase of cell cycle. It is characterized by a denser staining of the nuclear membrane, coalescence of chromatin into several pyknotic bodies, and eventual extrusion of the nuclear mass, leaving a cytoplasmic ghost. The sequential steps in degeneration may often be traced from one end of a synctial chain to the other, suggesting that the process may start with just one cell and then spread via intercellular bridges to involve all spermatogonia within the clone. Quanitatively, degeneration is a relatively constant feature of spermatogonial development. Only 25% of the theoretically possible number of pre-leptotene spermatocytes are produced from th original population of A1 spermatogonia; most of this loss is incurred during the maturation of A2 and A3 generations. While the reason for spermatogonial degeneration in the normal generminal epithelium remain obscure, it is proposed that the numerical ratio of A spermatogonia to Sertoli cells may be a significant limiting factor.

Animals↗

PubMed1979

Studies on the clonal origin of multiple myeloma. Use of individually specific (idiotype) antibodies to trace the oncogenic event to its earliest point of expression in B-cell differentiation.

IgA myeloma proteins of kappa- and lambda-types were isolated from two patients. These were used to produce and purify anti-idiotype antibodies of both broad (myeloma-related) and narrow (individual myeloma) specificities. The anti-idiotype antibodies were conjugated with fluorochromes and used as immunofluorescent probes to trace in the patients clonal expansion at different levels of B-cell differentiation. Our results (a) confirm that B lymphocyte precursors in IgA plasma-cell myelomas are involved in the malignant process, (b) show that B lymphocytes of the malignant clone include those expressing each of the major heavy-chain isotypes, mu, delta, gamma, and alpha, and (c) provide strong circumstantial evidence that pre-B-cell members of the malignant clone are also increased in frequency. T cells expressing idiotypic determinants were not detected. These findings argue that the initial oncogenic event may occur in a B-stem cell and is not influenced through stimulation by antigen. An interesting association was the increased frequency of related clones of B lymphocytes as detected by their reactivity with anti-idiotype antibodies of broad specificity. Neither plasma cell nor pre-B-cell members of these related clones were increased in frequency. Anti-idiotype antibodies or helper T cells reactive with myeloma-related idiotypes could be responsible for this phenomenon. We discuss other implications of these findings and speculate that all of the various phenotypes of B-lineage malignancies may result from oncogenic processes affecting stem cell targets.

Antibodies, Neoplasm↗

PubMed1979

Determinants of spontaneous recovery and persistance in MDCK cells infected with lymphocytic choriomeningitis virus.

MDCK cells that normally would have been killed by standard lymphocytic choriomeningitis (LCM) virus were saved either by pre- or co-infection with defective interfering (DI) virus. The ability of these spared cells to produce virus-specific antigen (as well as infectious virus) and resist being killed by standard virus challenge was followed for at least 35 days. During this period both types of cultures displayed unique cycling patterns for the above characteristics. The most striking difference was the longevity of the infections. Cultures exposed to DI particles prior to standard virus became persistently infected, while co-infection with both virus types led to spontaneous curing with no trace of the previous infection. The basis for these dissimilar outcomes was traced to a hitherto undetected non-defective LCM virus (called SP) in the DI virus stocks used to preinfect MDCK cells. SP virus was not present in standard virus stocks but arose in long-term persistently infected L cells that had been initially infected with standard virus. Cloned SP virus shared species-specific antigens with standard virus, was resistant to inhibition by DI virus and was capable of turning self-curing cultures into cultures persistently synthesizing both DI and SP virus.

Animals↗

PubMed1975

A live vaccine against tick-borne encephalitis: integrated studies. I. Basic properties and behaviour of the E5 "14" Clone (Langat virus).

The course of plaque segregation from the Langat E5 strain of the E5 "14" clone is described. The virus, displaying an ic+ sc s plus or minus t e u s character, reproduced at 35 and 37, but not at 39 degree C. In subcutaneously (sc) inoculated monkeys, viraemia lasted for 2--4 days without detectable central nervous system (CNS) involvement. In sc inoculated 8--10 g mice, the infection was manifested only by a transient trace viraemia, but caused a marked resistance against challenge with virulent tick-borne encephalitis (TE) viruses, protection indices being 5.7--6.7. Pathogenetic investigations in challenged, live virus-immunized mice showed no signs of a marked productive infection except of a booster effect.

Animals↗

PubMed1979

Antibody response to embryonal carcinoma cells in syngeneic mice.

The immunoglobulin (Ig) classes and subclasses of the specific antibodies contained in antisera raised in male 129/Sv mice against cells of a syngeneic clonal line of embryonal carcinoma (F9) have been determined. Cytotoxic activity was found associated almost exclusively with anti-F9 IgM (micro kappa) antibodies. A large part of anti-F9 activity was found associated with IgG1 (gamma 1 kappa and presumably also gamma 1 lambda) antibodies, and was detectable only by direct immunofluorescence. Traces of specific IgG2a and IgG2b antibodies were also found. No IgG3 and IgA antibodies reacting with embryonal carcinoma cells were detectable under these conditions. The serum of F9 tumor-bearing animals had a very similar Ig composition. Furthermore, IgM and IgG1, but not IgG2 antibodies, were detected at the surface of in vivo growing F9 tumor cells.

Animals↗

PubMed1978

Non-secretory or low-secretory myeloma with intracellular kappa chains. Report of six cases and review of the literature.

This report concerns six cases of multiple myeloma characterized by either no demonstrable monoclonal immunoglobulin in plasma or urine or by trace amounts (less than or equal to 0.1 g/l) of monoclonal kappa chains in the urine. In all cases there was an infiltration of the bone marrow by plasma cells containing kappa chains but no heavy chains. A retrospective analysis was made of 126 consecutive cases of Bence Jones myeloma. The number of kappa and lambda cases was approximately the same. All cases secreting less than or equal to 0.1 g light chains per 1 urine were of kappa type. This contrasts with a kappa/lambda ratio of 1.4-1.9 among reported series of M-components containing both heavy and light chains. A review of reported cases of non-secretory myeloma revealed a preserved capacity for Ig synthesis in the majority of cases and among these a preponderance of kappa chain producing clones. These observations might be explained by a higher tendency for kappa chain producing cells to mutate to low secretors or to cells producing abnormal light chains which are catabolized rapidly. The clinical data from our patients do not indicate a more pessimistic prognosis in non- or low-secretory myeloma, than in other cases of multiple myeloma.

Aged↗

PubMed1978

Variable region genes for the immunoglobulin framework are assembled from small segments of DNA--a hypothesis.

Sequences of each of the four framework segments FR1, FR2, FR3, and FR4 of the variable regions (V-regions) of light and heavy chains of immunoglobulins were grouped into sets with identical sequences. Sets contained from 1 to 18 members. When each V-region was traced from one FR to the next, it was seen that members of the same set in FR1 could be associated with different sets in FR2, FR3, and FR4. This suggests that the framework for the light and heavy chain V-regions is assembled during embryonic development from sets of minigenes for each FR segment. FR4 from three sets of human V(k)I chains also contained members of V(k)II, V(k)III, and V(k)IV subgroups; one FR2 set contained eight rabbit V(k), one human V(k)IV, and four mouse V(k) and an FR4 set contained two human V(H)III and one mouse V(H)III, indicating substantial evolutionary preservation of these sequences and suggesting that the sets of minigenes are highly conserved in the germ line. The clone of Tonegawa et al. [Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, O. & Gilbert, W. (1978) Proc. Natl. Acad. Sci. USA 75, 1485-1489] could be a hybrid FR1 and FR3 coming from minigenes of MOPC 315 (a V(lambda)II) whereas FR2 would come from MOPC 104E (a V(lambda)I). That FR4 is not joined to the rest of the V-region in 12-day-old mouse embryo DNA is also in accord with this hypothesis. Mouse sperm DNA should be examined to establish whether the hypothesized minigenes are separated by intervening sequences and whether the complementarity-determining (hypervariable) regions or segments of the V-region are separated from the framework in genomic DNA. Sperm DNA from rabbits or other species could be used to search for minigene segments whose sequences are identical in several species.

Animals↗