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Results for “Extrachromosomal Inheritance”

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At least 379 records · Page 21Linked to original sources
PubMed1990

Polyoma DNA replication dependent upon growth condition of SEWA sarcoma cells.

Extrachromosomal replication of viral DNA sequences has been observed in transformed as well as in normal cells following "stress"-inducing treatments. To explore the effect of growth conditions on the ability to support such replication, we analyzed SEWA sarcoma cells that grew subcutaneously or as ascites tumors in vivo as well as cell lines that were established from each of these tumors. The replicative form of polyoma DNA sequences was observed in SEWA tumors grown in ascites fluids but not in cells maintained as solid tumors. Polyoma DNA replication was found in ascites-derived cells that were adapted to grow in culture, only when the cultured cells are stimulated with UV irradiation. Immunoprecipitation of T antigens enabled detection of large T antigen only in the ascites-derived cells. The mechanisms that may regulate this phenomenon and the possible role large T may play in different growth conditions of SEWA cells are discussed.

Animals↗
PubMed1992

Detection of extrachromosomal circular DNA sequences from tumor cells by an alkaline lysis, Alu-polymerase chain reaction technique.

Extrachromosomal circular DNAs ranging in size from submicroscopic molecules of approximately 100 kb to cytogenetically resolvable structures of 1000+ kb called minute and double-minute chromosomes have been shown to harbor amplified genes in primary tumor cells, tumor cell lines, and drug-resistant cells grown in vitro. The presence of these molecules in transformed and malignant cells trends to reflect genetic instability and also suggests that role in tumor progression. Using a colon carcinoma cell line, we developed a technique to detect extrachromosomal circular DNA-specific sequences by Alu-polymerase chain reaction. Circular DNA was enriched by selective alkaline denaturation of genomic DNA. We have successfully performed this procedure with a minimum of 5 x 10(5) cells. The technique does not require any prior knowledge of the sequences located on the covalent circular DNA molecules for their detection. The procedure should be useful as a routine screen of primary tumor cells for the presence of extrachromosomal circular DNA and should permit the preparation of specific probes ot aid in their detailed characterizations.

Base Sequence↗
PubMed2004

Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system.

Recently, small interfering RNAs (siRNAs) have become a powerful and widely used tool for the analysis of gene function in mammalian cells. Here we report that the microinjection of an siRNA expression vector into the nucleus is an efficient and powerful method of specific gene silencing in pre-implantation mouse embryos. We used this method to examine the expression of two genes EGFP and Oct4. Vectors encoding siRNAs targeted against EGFP or Oct4 were injected into the pronucleus or nucleus of zygotes, which were then cultured until the blastocyst stage. When the effects of RNAi were examined in blastocyst stage eggs, there was robust inhibition of the gene product in a concentration-dependent manner at both the mRNA and the protein level. The expression of other endogenous genes was not affected, showing the specificity of the vector-mediated RNAi. In addition, this method was effective for inhibiting maternally expressed mRNA. To demonstrate that RNAi of Oct4 induced a similar phenotype to that of Oct4-null embryos, the blastocysts were further cultured in ES medium. After the fourth day of culture, the embryos either had outgrown only a layer of trophoblast cells or showed developmental arrest at the blastocyst stage (>90%). Moreover, concomitant with Oct4 suppression at the blastocyst stage, we observed inhibition of Fgf4, a gene that is known to be induced downstream of Oct4 expression. Taken together, these results demonstrate that the use of siRNA expression vector is a powerful way to achieve gene silencing in the pre-implantation stage embryo.

Animals↗
PubMed2004

Obtaining mice that carry human mitochondrial DNA transmitted to the progeny.

To study human diseases associated with mutations in mitochondrial DNA one needs an animal model in which the distribution of abnormal mtDNA and its impact on the phenotype might be followed. We isolated human mitochondria from HepG2 cell culture and microinjected them into murine zygotes, upon which those were transplanted to the pseudopregnant mice. PCR with species-specific primers allowed detecting human mtDNA in the tissues of 7-13-day embryos. No serious alterations in the development of transmitochondrial embryos were noticed. Among various organs/tissues of the 13-day embryos, human mtDNA was detected only in the heart, skeletal muscles, and stomach, which is in line with its uneven distribution among the blastomeres of an early mouse embryo that we described previously. In four recipient females, the microinjected zygotes were allowed to develop to term, the four neonate males of their joint litter were sacrificed, and in three of them human mtDNA was detected in the heart, skeletal muscles, stomach, brain, testes, and bladder. Six females of that joint litter were grown and mated to intact males. In the progeny (F1) of one of the females two mice were carrying human mtDNA in the heart, skeletal muscles, stomach, brain, lungs, uterus, ovaries, and kidneys. The study confirms the possibility to obtain transmitochondrial mice carrying human mtDNA that is transmitted to the animals of the next generation. Our results also indicate that among the organs to which human mtDNA is distributed some are more likely to receive it than others.

Animals↗
PubMed2006

Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells.

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.

Animals↗