scholarly journals Regulation of a human cardiac actin gene introduced into rat L6 myoblasts suggests a defect in their myogenic program.

1986 ◽  
Vol 6 (9) ◽  
pp. 3287-3290 ◽  
Author(s):  
R Hickey ◽  
A Skoultchi ◽  
P Gunning ◽  
L Kedes
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Human Gene ◽  
Actin Gene ◽  
Cardiac Actin ◽  
Gene Transcripts ◽  
Actin Mrna ◽  
Muscle Gene ◽  
Alpha Actin ◽  
Myogenic Program

The rat myogenic cell line L6E9 induces skeletal but not cardiac alpha-actin mRNA upon fusion to form myotubes. However, when a human cardiac alpha-actin gene was introduced into L6E9 myoblasts, differentiation of the cells led to the accumulation of human gene transcripts in parallel with those derived from the endogenous skeletal alpha-actin gene. This result demonstrates that factors which direct rat myogenesis can regulate a muscle gene from another species and that the L6E9 cells may have a defect in their ability to activate endogenous cardiac actin gene expression.

Regulation of a human cardiac actin gene introduced into rat L6 myoblasts suggests a defect in their myogenic program

1986 ◽  
Vol 6 (9) ◽  
pp. 3287-3290
Author(s):  
R Hickey ◽  
A Skoultchi ◽  
P Gunning ◽  
L Kedes
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Human Gene ◽  
Actin Gene ◽  
Cardiac Actin ◽  
Gene Transcripts ◽  
Actin Mrna ◽  
Muscle Gene ◽  
Alpha Actin ◽  
Myogenic Program

The rat myogenic cell line L6E9 induces skeletal but not cardiac alpha-actin mRNA upon fusion to form myotubes. However, when a human cardiac alpha-actin gene was introduced into L6E9 myoblasts, differentiation of the cells led to the accumulation of human gene transcripts in parallel with those derived from the endogenous skeletal alpha-actin gene. This result demonstrates that factors which direct rat myogenesis can regulate a muscle gene from another species and that the L6E9 cells may have a defect in their ability to activate endogenous cardiac actin gene expression.

Regulated expression of a transfected human cardiac actin gene during differentiation of multipotential murine embryonal carcinoma cells

1988 ◽  
Vol 8 (1) ◽  
pp. 406-417
Author(s):  
M A Rudnicki ◽  
M Ruben ◽  
M W McBurney
Keyword(s):  
Cardiac Muscle ◽  
Thymidine Kinase ◽  
Embryonal Carcinoma ◽  
Actin Gene ◽  
P19 Cells ◽  
Cardiac Muscle Cells ◽  
Cardiac Actin ◽  
Ec Cells ◽  
Actin Promoter ◽  
Actin Mrna

P19 embryonal carcinoma (EC) cells are multipotential stem cells which can be induced to differentiate in vitro into a variety of cell types, including cardiac muscle cells. A cloned human cardiac actin (CH-actin) gene was transfected into P19 cells, and stable transformants were isolated. Low levels of CH-actin mRNA were present in transformed EC cells, but a marked increase in the level of CH-actin mRNA was found as these cells differentiated into cardiac muscle. The accumulation of CH-actin mRNA paralleled that of the endogenous mouse cardiac actin mRNA. A chimeric gene, which consisted of the CH-actin promoter linked to the herpes simplex virus thymidine kinase coding region, was constructed and transfected into P19 cells. In these transformants, the thymidine kinase protein was located almost exclusively in cardiac muscle cells and was generally not detectable in EC or other nonmuscle cells. These results suggest that the transfected CH-actin promoter functions in the appropriate developmental and tissue-specific manner during the differentiation of multipotential EC cells in culture.

Muscle gene activation by induction and the nonrequirement for cell division

Development ◽  
1986 ◽  
Vol 97 (Supplement) ◽  
pp. 75-84
Author(s):  
J. B. Gurdon ◽  
S. Fairman
Keyword(s):  
Gene Activation ◽  
Cell Types ◽  
Northern Analysis ◽  
Muscle Actin ◽  
S1 Nuclease ◽  
Cardiac Actin ◽  
Gene Transcripts ◽  
Cloned Cdna ◽  
Muscle Gene ◽  
Neurula Stage

In amphibia, as in many other animals with free-swimming larvae, muscle is one of the first differentiated cell types to be formed in early development. In Xenopus, the first contractions of axial body muscle take place about 30 h after fertilization, but genes required to form muscle are activated long before this, during gastrulation. Muscle actin proteins are first seen to be synthesized at the early neurula stage (Sturgess et al. 1980). More recently Mohun et al. (1984), using cloned cDNA probes, have found that cardiac actin, the type of muscle actin characteristic of adult heart, is a major component of the larval axial muscle. Xenopus cardiac actin gene transcripts are detected by S1 nuclease and Northern analysis at the early neurula stage (Mohun et al. 1984), and the use of SP6 probes on poly(A)+ RNA enables cardiac actin transcripts to be seen as early as the midgastrula stage (Cascio & Gurdon, 1986).

Human gene expression microarray analysis of the HPV 6bE7-HaCaT stable cell line

Gene ◽  
2018 ◽  
Vol 657 ◽  
pp. 60-68 ◽  
Author(s):  
Boya Zhang ◽  
Xianzhen Chen ◽  
Qiang Zhou ◽  
Yinjing Song ◽  
Siyuan Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Microarray Analysis ◽  
Human Gene ◽  
Stable Cell Line ◽  
Gene Expression Microarray ◽  
Expression Microarray ◽  
Human Gene Expression ◽  
Gene Expression Microarray Analysis

scholarly journals Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression.

1988 ◽  
Vol 106 (6) ◽  
pp. 2077-2086 ◽  
Author(s):  
L J Hayward ◽  
Y Y Zhu ◽  
R J Schwartz
Keyword(s):  
Cellular Localization ◽  
Myoblast Fusion ◽  
Bipolar Cells ◽  
Gene Probe ◽  
Actin Gene ◽  
Cardiac Actin ◽  
Beta Actin ◽  
Hybridization Probes ◽  
Muscle Cultures ◽  
Actin Mrna

Specific DNA fragments complementary to the 3' untranslated regions of the beta-, alpha-cardiac, and alpha-skeletal actin mRNAs were used as in situ hybridization probes to examine differential expression and distribution of these mRNAs in primary myogenic cultures. We demonstrated that prefusion bipolar-shaped cells derived from day 3 dissociated embryonic somites were equivalent to myoblasts derived from embryonic day 11-12 pectoral tissue with respect to the expression of the alpha-cardiac actin gene. Fibroblasts present in primary muscle cultures were not labeled by the alpha-cardiac actin gene probe. Since virtually all of the bipolar cells express alpha-cardiac actin mRNA before fusion, we suggest that the bipolar phenotype may distinguish a committed myogenic cell type. In contrast, alpha-skeletal actin mRNA accumulates only in multinucleated myotubes and appears to be regulated independently from the alpha-cardiac actin gene. Accumulation of alpha-skeletal but not alpha-cardiac actin mRNA can be blocked by growth in Ca2+-deficient medium which arrests myoblast fusion. Thus, the sequential appearance of alpha-cardiac and then alpha-skeletal actin mRNA may result from factors that arise during terminal differentiation. Finally, the beta-actin mRNA was located in both fibroblasts and myoblasts but diminished in content during myoblast fusion and was absent from differentiated myotubes. It appears that in primary myogenic cultures, an asynchronous stage-dependent induction of two different alpha-striated actin mRNA species occurs concomitant with the deinduction of the nonmuscle beta-actin gene.

Differential expression of the myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis

1994 ◽  
Vol 14 (8) ◽  
pp. 5130-5138
Author(s):  
S Goswami ◽  
P Qasba ◽  
S Ghatpande ◽  
S Carleton ◽  
A K Deshpande ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cardiac Muscle ◽  
Differential Expression ◽  
Actin Gene ◽  
Myocyte Enhancer Factor 2 ◽  
Tissue Specific ◽  
Myocyte Enhancer Factor ◽  
Gene Transcripts ◽  
Alpha Actin ◽  
Enhancer Factor

In the present study, we have used single chicken blastoderms of defined early developmental stages, beginning with the prestreak stage, stage 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the functional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i.e., immediately after the cardiogenic fate assignment at stage 4. Consistent with this observation is the developmental expression pattern of DNA-binding activity of BBF-1, a cardiac muscle-specific member of the MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed with another AT-rich DNA sequence (CArG box) as probe, but the binding pattern with the ubiquitous TATA-binding proteins remains unchanged during the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appearance of cardiac MEF-2, MLC2, and alpha-actin gene products, occur earlier than previously thought and appear to be closely linked. The onset of skeletal myogenic program follows that of the cardiogenic program with the appearance of skeletal MLC2 at stage 8. We also observed that mRNA for the MEF-2 family of proteins appears as early as stage 2 and that for CMD-1, the chicken counterpart of MyoD, appears at stage 5. The temporal separation of activation of cardiac and skeletal MLC2 genes, which appears immediately after the respective fate assignments, and those of cardiac MEF-2 and CMD-1, which occur before, are consistent with the established appearance of the myogenic programs and with the acquisition pattern of the two tissue-specific morphological characteristics in the early embryo. The preferential appearance of BBF-1 activity in precardiac moesderm, relative to that of MEF-2, indicates that these two protein factors are distinct members of the MEF-2 family and provides a compelling argument in support of the potential role of BBF-1 as a regulator of the cardiogenic cell lineage determination, while cardiac MEF-2 might be involved in maintenance of the cardiac differentiative state.

Microvesicles Transcripts As Hallmark and Vector From Leukemic Parental Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1459-1459
Author(s):  
Gloria Milani ◽  
Tobia Lana ◽  
Silvia Bresolin ◽  
Francesca Paderi ◽  
Chiara Frasson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
K562 Cells ◽  
Parental Cell ◽  
Fold Change ◽  
Leukemia Cell Line ◽  
Target Cells ◽  
Rna Quality ◽  
Gene Transcripts ◽  
Selection Of

Abstract Abstract 1459 Microvesicles (MVs) are nano-sized lipid bodies (100–1000 nm in diameter) that are released by cells, both in vitro and in vivo. MVs are secreted in the extracellular space and as carriers of proteins, mRNA and miRNA represent vectors from donor to target cells involved in intercellular communication. MVs modulate the functional state of receiving cells through fusion with the target cell. In leukemia MVs were suggested to modulate the hematopoietic niche. In this study, we investigated the transcriptome of MVs released from leukemic cell lines. In particular, we analyzed K562, a BCR-ABL positive human erythromyeloblastoid leukemia cell line, and we collected RNA both from cells and MVs released in culture. Since many different methods have been described for microvesicles isolation and description of MV populations are often ambiguous, an accurate protocol has been developed in order to select a defined MVs population. In detail, for MVs isolation cell culture medium was centrifuged at 2500g for 15 minutes. These centrifugations allowed to delete cells and bigger bodies. Then supernatant was filtered by means of a 1.2um filter, in order to keep only vesicles of defined physical measure and to eliminate residual bigger vesicles, such as apoptotic bodies (>1000nm). The filtration allowed an accurate selection of a well defined MVs population. The filtered medium was then centrifuged at 18000g for 1h at 4°C. MVs were resuspended in Trizol for RNA isolation. Also RNA of cells, from which MVs have been released, was extracted using the Trizol method, according to manufacturer instructions. Cell line RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by means of NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). To check MVs RNA integrity we determined the ratio of 5' amplicons to 3' amplicons of housekeeping transcripts by Real Time PCR. We screened the presence of several housekeeping transcripts in MVs and selected GAPDH as reference. The GAPDH 5':3' amplicon ratio should equal 1 to ensure MVs RNA quality. Afterwards, reverse transcription–polymerase chain reaction (RT-PCR) amplification was performed. cDNA was synthesized from 1mg of total RNA. We analyzed the gene expression profile of K562 cell line and MVs, released from these cells, using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). Gene expression data have been compared between cells and MVs. Ninety-one percent of probe sets showed a similar expression (fold-change lower than 1.5) between cell line and MVs. Thirteen percent of these probes were recognized as present call probe sets in both cell line and MVs. Analysis of probe sets using DAVID Functional Annotation Bioinformatics Microarray Analysis revealed a conservation of MVs gene transcripts involved in pathways of cell function such as RNA processing, protein translation, aminoacid metabolism and cell respiration. Remarkably, in MVs we observed a high presence of gene transcripts coding for protein belonging to the Chronic Myeloid Leukemia pathway that are expressed downstream of the BCR-ABL tyrosin kinase fusion protein. The maintenance of this pathway in MVs highlights the intrinsic peculiarity of BCR-ABL positive K562 cells apparently also conserved in MVs mRNA outfit representing a hallmark of the parental cell from which they have been released. Furthermore, 3.8% of the probe sets resulted to be up-regulated in MVs compare to the cell line (fold-change higher than 1.5). In MVs, we observed a higher expression of genes belonging to cell communication pathways, adhesion and migration processes, membrane and ionic channels signals. In conclusion, we isolated MVs released by K562 leukemic cells using an accurate selection of the MV population based on physical measures and for the first time a whole transcriptome gene expression analysis has been performed comparing K562 cells and MVs. Moreover, we identified an enrichment of transcripts coding for proteins involved in several essential pathways in the MVs supporting the hypothesis of a functional selection from the parental cell transcriptome and underlining the relevant role of MVs as vehicles of messages to target cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulated expression of a transfected human cardiac actin gene during differentiation of multipotential murine embryonal carcinoma cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 406-417 ◽  
Author(s):  
M A Rudnicki ◽  
M Ruben ◽  
M W McBurney
Keyword(s):  
Cardiac Muscle ◽  
Thymidine Kinase ◽  
Embryonal Carcinoma ◽  
Actin Gene ◽  
P19 Cells ◽  
Cardiac Muscle Cells ◽  
Cardiac Actin ◽  
Ec Cells ◽  
Actin Promoter ◽  
Actin Mrna

P19 embryonal carcinoma (EC) cells are multipotential stem cells which can be induced to differentiate in vitro into a variety of cell types, including cardiac muscle cells. A cloned human cardiac actin (CH-actin) gene was transfected into P19 cells, and stable transformants were isolated. Low levels of CH-actin mRNA were present in transformed EC cells, but a marked increase in the level of CH-actin mRNA was found as these cells differentiated into cardiac muscle. The accumulation of CH-actin mRNA paralleled that of the endogenous mouse cardiac actin mRNA. A chimeric gene, which consisted of the CH-actin promoter linked to the herpes simplex virus thymidine kinase coding region, was constructed and transfected into P19 cells. In these transformants, the thymidine kinase protein was located almost exclusively in cardiac muscle cells and was generally not detectable in EC or other nonmuscle cells. These results suggest that the transfected CH-actin promoter functions in the appropriate developmental and tissue-specific manner during the differentiation of multipotential EC cells in culture.

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