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Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette.

Kononenko AV, Lee NC, Liskovykh M, Masumoto H, Earnshaw WC, Larionov V, Kouprina N - Nucleic Acids Res. (2015)

Bottom Line: Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step.We demonstrated that a single copy of tTA(VP64) carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function.To adopt the alphoid(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA(VP64) cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutic Branch, National Cancer Institute, Bethesda, MD 20892, USA.

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Kinetics of mitotic stability of the HAC carrying the construct tetR-VP64-IRES-DsRed2. The cells were cultured under following conditions: dox+ 3 days and dox− 3 days, dox+ 4 days and dox− 4 days, dox+ 7 days and dox− 7 days, dox+ 14 days and dox− 14 days. To quantify the HAC retention, the cells were analyzed by FISH. FISH analysis included detection of the HAC on metaphase spreads. At least 70–80 metaphase spreads were analyzed for each point. Addition of doxycycline, which prevents fusion of the tetracycline repressor from binding to tetO sequences of the HAC, blocks the inactivation of HAC kinetochore. Targeting the transcriptional transactivator tTAVP64 into the HAC kinetochore in the absence of doxycycline induces HAC loss.
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Figure 3: Kinetics of mitotic stability of the HAC carrying the construct tetR-VP64-IRES-DsRed2. The cells were cultured under following conditions: dox+ 3 days and dox− 3 days, dox+ 4 days and dox− 4 days, dox+ 7 days and dox− 7 days, dox+ 14 days and dox− 14 days. To quantify the HAC retention, the cells were analyzed by FISH. FISH analysis included detection of the HAC on metaphase spreads. At least 70–80 metaphase spreads were analyzed for each point. Addition of doxycycline, which prevents fusion of the tetracycline repressor from binding to tetO sequences of the HAC, blocks the inactivation of HAC kinetochore. Targeting the transcriptional transactivator tTAVP64 into the HAC kinetochore in the absence of doxycycline induces HAC loss.

Mentions: Next, we examined the kinetics of HAC loss in one of HT1080 clones after 3, 4, 7 and 14 days of culture in the medium with and without doxycycline. As seen, the fraction of cells carrying the HAC decreased dramatically in dox− medium after 4 days of culture while remaining relatively stable in dox+ medium (Figure 3).


Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette.

Kononenko AV, Lee NC, Liskovykh M, Masumoto H, Earnshaw WC, Larionov V, Kouprina N - Nucleic Acids Res. (2015)

Kinetics of mitotic stability of the HAC carrying the construct tetR-VP64-IRES-DsRed2. The cells were cultured under following conditions: dox+ 3 days and dox− 3 days, dox+ 4 days and dox− 4 days, dox+ 7 days and dox− 7 days, dox+ 14 days and dox− 14 days. To quantify the HAC retention, the cells were analyzed by FISH. FISH analysis included detection of the HAC on metaphase spreads. At least 70–80 metaphase spreads were analyzed for each point. Addition of doxycycline, which prevents fusion of the tetracycline repressor from binding to tetO sequences of the HAC, blocks the inactivation of HAC kinetochore. Targeting the transcriptional transactivator tTAVP64 into the HAC kinetochore in the absence of doxycycline induces HAC loss.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4482055&req=5

Figure 3: Kinetics of mitotic stability of the HAC carrying the construct tetR-VP64-IRES-DsRed2. The cells were cultured under following conditions: dox+ 3 days and dox− 3 days, dox+ 4 days and dox− 4 days, dox+ 7 days and dox− 7 days, dox+ 14 days and dox− 14 days. To quantify the HAC retention, the cells were analyzed by FISH. FISH analysis included detection of the HAC on metaphase spreads. At least 70–80 metaphase spreads were analyzed for each point. Addition of doxycycline, which prevents fusion of the tetracycline repressor from binding to tetO sequences of the HAC, blocks the inactivation of HAC kinetochore. Targeting the transcriptional transactivator tTAVP64 into the HAC kinetochore in the absence of doxycycline induces HAC loss.
Mentions: Next, we examined the kinetics of HAC loss in one of HT1080 clones after 3, 4, 7 and 14 days of culture in the medium with and without doxycycline. As seen, the fraction of cells carrying the HAC decreased dramatically in dox− medium after 4 days of culture while remaining relatively stable in dox+ medium (Figure 3).

Bottom Line: Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step.We demonstrated that a single copy of tTA(VP64) carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function.To adopt the alphoid(tetO)-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA(VP64) cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid(tetO)-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.

View Article: PubMed Central - PubMed

Affiliation: Developmental Therapeutic Branch, National Cancer Institute, Bethesda, MD 20892, USA.

Show MeSH
Related in: MedlinePlus