scholarly journals Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3251-3259 ◽  
Author(s):  
K Bhat ◽  
M W McBurney ◽  
H Hamada
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Regulatory Elements ◽  
Embryonal Carcinoma Cell ◽  
P19 Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Cellular Dna ◽  
Chromosomal Loci

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.

Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells

1988 ◽  
Vol 8 (8) ◽  
pp. 3251-3259
Author(s):  
K Bhat ◽  
M W McBurney ◽  
H Hamada
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Regulatory Elements ◽  
Embryonal Carcinoma Cell ◽  
P19 Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Cellular Dna ◽  
Chromosomal Loci

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.

scholarly journals Regulated expression of the small nuclear ribonucleoprotein particle protein SmN in embryonic stem cell differentiation.

1990 ◽  
Vol 10 (12) ◽  
pp. 6817-6820 ◽  
Author(s):  
N G Sharpe ◽  
D G Williams ◽  
D S Latchman
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Embryonal Carcinoma Cell ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The SmN protein is a component of small nuclear ribonucleoprotein particles and is closely related to the ubiquitous SmB and B' splicing proteins. It is expressed in a limited range of tissues and cell types, including several undifferentiated embryonal carcinoma cell lines and undifferentiated embryonic stem cells. The protein declines to undetectable levels when embryonal carcinoma or embryonic stem cells are induced to differentiate, producing primitive endoderm or parietal endoderm or yielding embryonal bodies. This decline is due to a corresponding decrease in the level of the SmN mRNA. The potential role of SmN in the regulation of alternative splicing in embryonic cell lines and early embryos is discussed.

Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome

1990 ◽  
Vol 10 (8) ◽  
pp. 4163-4169
Author(s):  
W J Pavan ◽  
P Hieter ◽  
R H Reeves
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Embryonal Carcinoma ◽  
Yeast Artificial Chromosome ◽  
Carcinoma Cell ◽  
Artificial Chromosome ◽  
Carcinoma Cell Line ◽  
Yeast Genome ◽  
Embryonal Carcinoma Cell ◽  
Embryonal Carcinoma Cell Line

A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.

scholarly journals Lineage-specific transformation after differentiation of multipotential murine stem cells containing a human oncogene.

1986 ◽  
Vol 6 (2) ◽  
pp. 617-625 ◽  
Author(s):  
J C Bell ◽  
K Jardine ◽  
M W McBurney
Keyword(s):  
Embryonal Carcinoma ◽  
Carcinoma Cell ◽  
Cell Lineage ◽  
Cell Types ◽  
Embryonal Carcinoma Cell ◽  
Fibroblast Cells ◽  
P19 Cells ◽  
Differentiated Cells ◽  
Embryonal Carcinoma Cell Line ◽  
Low Levels

We transfected the human EJ bladder carcinoma oncogene (Ha-rasEJ-1) into multipotential embryonal carcinoma cell line P19. The transgenic P19(ras+) cells expressed high levels of both the mRNA and the p21EJ protein derived from the oncogene. When cultured in the presence of retinoic acid, P19(ras+) cells differentiated and developed into the same spectrum of differentiated cell types as the parental P19 cells (namely, neurons, astrocytes, and fibroblast-like cells). Thus, it seems unlikely that the Ha-ras-1 proto-oncogene product plays a role in initiation of differentiation or in the choice of differentiated cell lineage. Most of the P19(ras+)-derived differentiated cells contained relatively low levels of p21EJ and were nontransformed, whereas certain cells with fibroblast-like morphology continued to express the Ha-rasEJ-1 gene at high levels and were transformed (i.e., immortal and anchorage independent). Fibroblasts derived from P19 cells did not become transformed following transfection of the Ha-rasEJ-1 oncogene, suggesting that transformation of the fibroblast cells only occurred if the oncogene was present and expressed during the early stages of the developmental lineage.

Regulated expression of the small nuclear ribonucleoprotein particle protein SmN in embryonic stem cell differentiation

1990 ◽  
Vol 10 (12) ◽  
pp. 6817-6820
Author(s):  
N G Sharpe ◽  
D G Williams ◽  
D S Latchman
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Embryonal Carcinoma Cell ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The SmN protein is a component of small nuclear ribonucleoprotein particles and is closely related to the ubiquitous SmB and B' splicing proteins. It is expressed in a limited range of tissues and cell types, including several undifferentiated embryonal carcinoma cell lines and undifferentiated embryonic stem cells. The protein declines to undetectable levels when embryonal carcinoma or embryonic stem cells are induced to differentiate, producing primitive endoderm or parietal endoderm or yielding embryonal bodies. This decline is due to a corresponding decrease in the level of the SmN mRNA. The potential role of SmN in the regulation of alternative splicing in embryonic cell lines and early embryos is discussed.

X chromosome reactivation in mouse embryonal carcinoma cells

1985 ◽  
Vol 5 (10) ◽  
pp. 2705-2712
Author(s):  
G D Paterno ◽  
C N Adra ◽  
M W McBurney
Keyword(s):  
X Chromosome ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
S Phase ◽  
Dna Demethylation ◽  
Embryonal Carcinoma Cell ◽  
X Chromosomes ◽  
Embryonal Carcinoma Cell Line ◽  
Inactive X Chromosome

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

scholarly journals X chromosome reactivation in mouse embryonal carcinoma cells.

1985 ◽  
Vol 5 (10) ◽  
pp. 2705-2712 ◽  
Author(s):  
G D Paterno ◽  
C N Adra ◽  
M W McBurney
Keyword(s):  
X Chromosome ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
S Phase ◽  
Dna Demethylation ◽  
Embryonal Carcinoma Cell ◽  
X Chromosomes ◽  
Embryonal Carcinoma Cell Line ◽  
Inactive X Chromosome

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

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