Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines

2016 ◽  
Vol 32 (4) ◽  
pp. 1061-1068 ◽  
Author(s):  
Jorge Quiroz ◽  
Yung-Shyeng Tsao
Keyword(s):  
Statistical Analysis ◽  
Cell Lines ◽  
Manufacturing Cell ◽  
Limiting Dilution ◽  
Limiting Dilution Cloning

scholarly journals Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones.

1986 ◽  
Vol 103 (5) ◽  
pp. 2025-2034 ◽  
Author(s):  
O D Madsen ◽  
L I Larsson ◽  
J F Rehfeld ◽  
T W Schwartz ◽  
A Lernmark ◽  
...  
Keyword(s):  
Cell Lines ◽  
Islet Cell ◽  
Islet Cell Tumor ◽  
Cell Tumor ◽  
Cell Extracts ◽  
Islet Hormones ◽  
G2 Cells ◽  
Limiting Dilution ◽  
Limiting Dilution Cloning

A liver metastasis (MSL) with a remarkable in vitro proliferation potential has been identified in an NEDH rat carrying a transplantable x-ray-induced islet cell tumor. Two insulin-secreting cell lines, MSL-G and MSL-H, with doubling times of 3-5 d were established by repeated limiting dilution cloning. In vivo inoculation of MSL-G cells induced severe hypoglycemia caused by a small but highly heterogeneous tumor as revealed by immunocytochemistry. Whereas most cells stained for the islet hormones, insulin, glucagon, and somatostatin, clustered cells were discovered to contain cholecystokinin (CCK). Additional in vitro-limiting dilution cloning, followed by immunocytochemical characterization, clearly demonstrated the capacity of single cell clones to simultaneously express the same four hormones. Radioimmunoassays with a panel of site-specific antisera of culture supernatants and purified cell extracts showed the MSL-G2 cells to produce, store, and secrete readily detectable amounts of processed and unprocessed CCK. Gastrin was not detected while coexpression of glucagon and CCK were demonstrated. Mutant clones selected for resistance to 6-thioguanine (frequency, 2 X 10(-7] and checked for HAT (hypoxanthine, aminopterin, thymidine) sensitivity retained the capacity for multi-hormone expression. We propose that the MSL tumor contains pluripotent endocrine stem cells. The MSL tumor and the MSL-G2 cells in particular will allow studies of not only CCK biosynthesis and processing but also of mechanisms involved in tumor and islet cell differentiation.

Limiting dilution cloning of B cells from patients with multiple myeloma: Emergence of non-malignant B-cell lines

1989 ◽  
Vol 43 (4) ◽  
pp. 578-586 ◽  
Author(s):  
G. Clofent ◽  
B. Klein ◽  
T. Commes ◽  
C. Vincent ◽  
N. Ghanem ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Limiting Dilution ◽  
Limiting Dilution Cloning

scholarly journals A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

10.3791/3010 ◽  
2011 ◽  
Author(s):  
Shuangping Shi ◽  
Russ G.G. Condon ◽  
Liang Deng ◽  
Jason Saunders ◽  
Finn Hung ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Protein Therapeutics ◽  
Manufacturing Cell ◽  
Automated Platform

Murphy's law of limiting dilution cloning revisited

1990 ◽  
Vol 9 (12) ◽  
pp. 1541-1541 ◽  
Author(s):  
Robert Staszewski
Keyword(s):  
Limiting Dilution ◽  
Limiting Dilution Cloning

Generation of Acute Myeloid Leukemia (AML)-Specific Autologous CD4 and CD8 T Cell Lines by Limiting Dilution AML Dendritic Cell Culture.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2018-2018
Author(s):  
Rui-kun Zhong ◽  
Thomas A. Lane ◽  
Edward D. Ball
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Cd8 T Cell ◽  
Ifn Gamma ◽  
T Cell Lines ◽  
Limiting Dilution ◽  
Acute Myeloid

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure

Gene Expression Profiling Reveals a Crucial Role for C/EBPbeta in Proliferation Pathways of ALK+ ALCL Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2818-2818
Author(s):  
Irina Bonzheim ◽  
Martin Irmler ◽  
Natasa Anastasov ◽  
Margit Klier ◽  
Johannes Beckers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Statistical Analysis ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Expression Profiling ◽  
Leucine Zipper ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Large Cell

Abstract Introduction: ALK+ anaplastic large cell lymphomas (ALCL) overexpress C/EBPβ, as a consequence of NPM-ALK kinase activity. C/EBPβ is a leucine zipper transcription factor, which plays a major role in cellular differentiation, inflammation, proliferation and metabolism control. To determine the role of C/EBPβ in ALK+ ALCL transformation, and to identify its downstream targets, a highly specific C/EBPβ-shRNA was used to knockdown C/EBPβ. The consequences of C/EBPβ gene-silencing were analyzed by gene expression profiling. Materials and Methods: Four ALK+ ALCL cell lines, SUDHL-1, Kijk, Karpas 299 and SUP-M2 were transfected with lentivirus containing the C/EBPβ shRNA or the vector without shRNA in triplicates. Western Blot analysis and qRT-PCR were performed to quantify the knockdown effect. At day three after infection, RNA was extracted and used for Gene Chip expression analysis (Affymetrix). Using Anova software for statistical analysis, we identified genes, which were regulated in all four cell lines. The effect of C/EBPβ knockdown on proliferation, cell cycle, and viability was analyzed by MTT assay and FACS analysis. Results: In all four ALK+ ALCL, efficient C/EBPβ knockdown resulted in profound growth retardation (up to 84%) compared to control cells after 6 days of infection, and a clear shift from the S phase to the G1 phase in the cell cycle was observed. To identify genes regulated by C/EBPβ in all four cell lines, we performed statistical analysis applying a false discovery rate of 20%, and accepted only genes with a >1,1 and <0,9 fold ratio. We identfied 435 genes regulated after C/EBPβ knockdown (117 upregulated, 318 downregulated). Classification of the differentially expressed genes into biological categories revealed overrepresentation of genes involved in the regulation of kinase activity, cell cycle and proliferation, lymphocyte differentiation, and metabolic processes. In particular, kinases involved in the regulation of JNK activity, which have been shown previously to be involved in proliferation of ALCL, were highly affected by C/EBPβ knockdown. Genomatix Bibliosphere Pathway Analysis revealed C/EBPβ to be connected to pathways involving cell cycle (RUNX3, CCNG1, CDKN2A), apoptosis (FAS, PTPRC, BCL2A1, BIRC3) and MAPK cascades (TRIB1 and several MAP3Ks). Several of the genes identified contain known C/EBPβ binding sites. Conclusions: C/EBPβ silencing induces growth arrest in ALK+ALCL, which correlates with differential expression of genes involved in cell cycle, apoptosis and differentiation. This study reveals C/EBPβ as a master transcription regulator of NPM-ALK induced cellular proliferation, and therefore, an ideal candidate for targeted therapeutic intervention.

scholarly journals Array-Based Transcript Profiling and Limiting-Dilution Reverse Transcription-PCR Analysis Identify Additional Latent Genes in Kaposi's Sarcoma-Associated Herpesvirus

2010 ◽  
Vol 84 (11) ◽  
pp. 5565-5573 ◽  
Author(s):  
Sanjay Chandriani ◽  
Don Ganem
Keyword(s):  
Infected Cell ◽  
B Cell ◽  
Cell Lines ◽  
Reverse Transcription ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Transcript Profiling ◽  
Reverse Transcription Pcr ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Limiting Dilution

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a B-lymphotropic herpesvirus strongly linked to both lymphoproliferative diseases and Kaposi's sarcoma. The viral latency program of KSHV is central to persistent infection and plays important roles in the pathogenesis of KSHV-related tumors. Up to six polypeptides and 18 microRNAs are known to be expressed in latency, but it is unclear if all major latency genes have been identified. Here, we have employed array-based transcript profiling and limiting-dilution reverse transcription-PCR (RT-PCR) methodologies to explore this issue in several KSHV-infected cell lines. Our results show that RNAs encoding the K1 protein are found at low levels in most latently infected cell lines. The gene encoding v-IL-6 is also expressed as a latent transcript in some contexts. Both genes encode powerful signaling molecules with particular relevance to B cell biology: K1 mimics signaling through the B cell receptor, and v-IL-6 promotes B cell survival. These data resolve earlier controversies about K1 and v-IL-6 expression and indicate that, in addition to core latency genes, some transcripts can be expressed in KSHV latency in a context-dependent manner.

scholarly journals Limiting dilution comparison of the repertoires of high and low responder MHC-restricted T cells.

1988 ◽  
Vol 167 (3) ◽  
pp. 1100-1113 ◽  
Author(s):  
M Kojima ◽  
K B Cease ◽  
G K Buckenmeyer ◽  
J A Berzofsky
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Sperm Whale ◽  
Cell Repertoire ◽  
Restriction Element ◽  
Low Responder ◽  
Low X ◽  
Limiting Dilution ◽  
Immunodominant Epitopes

To approach the mechanism that determines Ir gene-controlled high or low responsiveness to whole proteins, such as sperm whale myoglobin (SWMb), we compared the repertoires of high and low responder haplotype-restricted T cells for different myoglobin epitopes by limiting dilution frequency analysis. Poisson analysis was performed using long-term limiting dilution cell lines of (B10.BR [low] X B10.D2[high])F1 T cells maintained on high or low responder APCs. The cell lines were tested with SWMb peptides and fragments for T cell repertoire fine specificities and Ia restrictions. The frequency of SWMb-specific F1 T cells responsive on B10.BR (H-2k) APCs was 2.5-3.6-fold lower than on B10.D2 (H-2d) APCs. Strikingly, all of the H-2k-restricted T cells used I-Ek as a restriction element, whereas both I-Ad- and I-Ed-restricted T cells were found among the H-2d-restricted lines. The I-Ad-restricted T cells were dominant, and the majority was specific for the synthetic peptide 102-118. T cells specific for peptide 132-146, dominant in association with I-Ed, were less frequent. However, no detectable H-2k-restricted T cells were specific for either of these peptides, but instead they were specific for fragment 1-55 or peptide 59-80. Fragment 1-55 also stimulated a similar number of H-2d-restricted T cells. Therefore, the low response of F1 T cells on H-2k-presenting cells may be due to the failure to see myoglobin plus I-Ak, in particular the immunodominant site around Glu 109, in contrast to the dominant response of high responder mice (both H-2d and H-2s) focused on the I-A molecule and the site around residue Glu 109. The I-E- low responder B10 strain also failed to respond to peptide 102-118, supporting the idea that the low responder status results from a limited repertoire lacking response to 102-118 plus I-A. In those strains that respond to the immunodominant site 102-118, the frequency of T cells in the repertoire specific for this site was always considerably greater than that for other sites. These results suggest that there is an important difference between immunodominant epitopes and minor epitopes and that Ir gene-controlled low responsiveness to a natural whole protein may be due primarily to the failure to respond to a single immunodominant site, even though a number of other epitopes can be recognized.

scholarly journals 231EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY

2004 ◽  
Vol 16 (2) ◽  
pp. 236 ◽  
Author(s):  
Z. Beyhan ◽  
N.L. First
Keyword(s):  
Gene Expression ◽  
Statistical Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Live Birth ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Gene Expression Patterns ◽  
Donor Cells

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.

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