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In JoVE (2)
- Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência
- Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA
Other Publications (6)
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Articles by Michelle L. Collins in JoVE
JoVE General
Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência
Gene Expression Division, Bio-Rad Laboratories, Inc.
Este procedimento mostra como usar o sistema de Gene Pulser eletroporação MXcell rapidamente e facilmente identificar as melhores condições de eletroporação de fibroblastos de rato embrionárias (MEFs) ou outras células primárias. Considerações para solução de problemas também são discutidos no vídeo associado.
JoVE General
Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA
Gene Expression Division, Bio-Rad Laboratories
Este procedimento descreve um fluxo de trabalho rápido e fácil de introduzir em siRNA difícil de transfecção de linhas celulares e siga expressão gênica por PCR em tempo real. Uso de um contador de células automatizado, multi-prato bem eletroporação, e da estação de eletroforese automatizada fornecer resultados rápidos e confiáveis, sem a necessidade de manipulação robótica caro.
Other articles by Michelle L. Collins on PubMed
Measurement of Mitochondrial DNA Synthesis in Vivo Using a Stable Isotope-mass Spectrometric Technique
We describe here a new stable isotope-mass spectrometric technique for measuring mitochondrial DNA (mtDNA) synthesis. Growing (2-4 mo old) and weight-stable (8-10 mo old) Sprague-Dawley rats were primed with (2)H(2)O (deuterated water) to 2.0-2.5% body water enrichment, via intraperitoneal injection, and then given 4% (2)H(2)O in drinking water for 3-11 wk. Mitochondria were isolated from cardiac and hindlimb muscle, and mtDNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides. PCR confirmed the absence of nuclear DNA contamination. The isotopic enrichment of the deoxyribose moiety of deoxyadenosine was determined by GC-MS analysis, and percent new mtDNA was calculated by comparison to genomic DNA enrichments in a tissue with nearly complete turnover (bone marrow). Initial label incorporation into deoxyadenosine of mtDNA was linear, and turnover of mtDNA was observed in nongrowing adult female rats (1.1-1.3% new mtDNA per day in cardiac and skeletal muscle). Die-away curves of mtDNA after discontinuing (2)H(2)O administration gave a similar turnover rate constant. Human subjects were also given (2)H(2)O for up to 6 wk, and mitochondria from platelets were isolated. Incubation with DNase removed any contaminating genomic DNA; platelet mtDNA exhibited linear incorporation from (2)H(2)O and reached plateau values identical to those in genomic DNA from fully turned over cells (circulating monocytes). In conclusion, replication of mtDNA can be directly measured in vivo in rodents and humans without the use of radioactivity. Use of this technique may allow improved understanding of the regulation of mitochondrial biogenesis in health and disease.
Effect of Nucleoside Reverse Transcriptase Inhibitors on Mitochondrial DNA Synthesis in Rats and Humans
Nucleoside reverse transcriptase inhibitors (NRTIs) have been hypothesized to inhibit mitochondrial DNA polymerase gamma, resulting in decreased mtDNA synthesis and mitochondrial insufficiency in HIV-1-infected patients. mtDNA synthesis was measured directly using a stable isotope mass spectrometric method following NRTI treatment in rodents. 3'-Azido-3'-deoxythymidine (AZT) was added to water (1 mg/mL) and administered ad libitum to female Sprague-Dawley rats for 1-8 weeks (n = 4 or 5 animals/timepoint). Neither body weight nor food intake was affected by AZT intake. Untreated controls and AZT-treated rats were given 4% H2O as drinking water for 2 weeks. AZT (approximately 100 mg/kg/d) produced a significant (P < 0.05) decrease in cardiac and hindlimb muscle mtDNA fractional synthesis compared with control groups (from 13.8 +/- 4.2% to 7.0 +/- 4.8% and from 7.6 +/- 1.8% to 4.5 +/- 0.4%, respectively) after 4 weeks. Cytochrome c oxidase content in hindlimb muscle was also decreased by 50% compared with controls after 4 weeks of AZT treatment (P < 0.07) and a calculated index of absolute mitochondrial biogenesis rate was significantly reduced by week 2 of AZT (P < 0.05) in hindlimb muscle. In preliminary studies, platelet mtDNA enrichments were compared to monocyte nDNA enrichments (used as a marker of a fully turned over tissue) in healthy human subjects. Fractional synthesis of mtDNA in platelets reached 98 +/- 3% after 5 weeks of H2O labeling. It is concluded that NRTIs decrease mtDNA synthesis and oxidative enzyme content and thus mitochondrial biogenesis in rodents and that the effects of NRTIs on mitochondrial biogenesis in tissues of HIV-1- infected humans can in principle be measured using this approach.
Characterisation of Complex Formation Between Members of the Mycobacterium Tuberculosis Complex CFP-10/ESAT-6 Protein Family: Towards an Understanding of the Rules Governing Complex Formation and Thereby Functional Flexibility
We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.
Creation of a Stepdown Nurse Internship Program
Within Christiana Care Health System, an opportunity evolved to develop a nurse recruitment strategy for our stepdown units. Being a large teaching system, there was specific need for nursing education at this entry level. Through collaboration of nursing colleagues, the Stepdown Nurse Internship Program was created. The program encompassed clinical orientation and didactic classes. Of notable significance at the completion of the program were the Basic Knowledge Assessment Test score improvement and the nurse retention rate.
The Remnants of Galaxy Formation from a Panoramic Survey of the Region Around M31
In hierarchical cosmological models, galaxies grow in mass through the continual accretion of smaller ones. The tidal disruption of these systems is expected to result in loosely bound stars surrounding the galaxy, at distances that reach 10-100 times the radius of the central disk. The number, luminosity and morphology of the relics of this process provide significant clues to galaxy formation history, but obtaining a comprehensive survey of these components is difficult because of their intrinsic faintness and vast extent. Here we report a panoramic survey of the Andromeda galaxy (M31). We detect stars and coherent structures that are almost certainly remnants of dwarf galaxies destroyed by the tidal field of M31. An improved census of their surviving counterparts implies that three-quarters of M31's satellites brighter than M(v) = -6 await discovery. The brightest companion, Triangulum (M33), is surrounded by a stellar structure that provides persuasive evidence for a recent encounter with M31. This panorama of galaxy structure directly confirms the basic tenets of the hierarchical galaxy formation model and reveals the shared history of M31 and M33 in the unceasing build-up of galaxies.
