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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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(a) A scheme representing binding of tTAVP64 protein with the HAC followed by HAC loss from the cells as result of doxycycline removal. (b) Bright and fluorescent images of one representative clone with the HAC carrying the tTAVP64-DsRed2 cassette cultured in dox+ medium and after removal doxycycline followed by 14 days of culture. Cells were cultured without HAC selection (in HAT− and BS− medium). (c) Representative images of the metaphase spread after 14 days of culture in the presence and absence of doxycycline. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (red).
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Figure 2: (a) A scheme representing binding of tTAVP64 protein with the HAC followed by HAC loss from the cells as result of doxycycline removal. (b) Bright and fluorescent images of one representative clone with the HAC carrying the tTAVP64-DsRed2 cassette cultured in dox+ medium and after removal doxycycline followed by 14 days of culture. Cells were cultured without HAC selection (in HAT− and BS− medium). (c) Representative images of the metaphase spread after 14 days of culture in the presence and absence of doxycycline. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (red).

Mentions: In contrast, a dramatic alphoidtetO HAC loss was observed in dox− medium when the tTAVP64 cassette was inserted into the HAC. Data on FISH analysis presented as a percentage of HAC-containing metaphases in different media (dox+ versus dox−) are summarized in Table 1. As seen, the absence or presence of doxycycline had essentially little, if any, effect on stability of the HAC after 3 days of culture. However, after 14 days, expression of the tTAVP64 fusion protein caused a dramatic increase in the population of cells lacking the HAC when the cells were grown in the absence of doxycycline (dox−). As a consequence, a disappearance of the DsRed2 fluorescence signal expressed from the HAC was observed when the cells were grown in the absence of doxycycline (Figure 2a and b). Note that expression of a single copy tTAVP64 from the HAC is 57-times lower than expression of the same transgene from the multiple copy vector transiently transfected into HT1080 cells (Supplementary Figure S4). Differences between two analyzed tTA cassettes is likely explained by a stronger effect of four tandem repeats of the VP16 domain in tTAVP64 on chromatin status in the HAC kinetochore compared to tTAVP16. Indeed, tTAVP64 was developed to increase the potency of transcriptional activation of tTAVP16 (36).


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)

(a) A scheme representing binding of tTAVP64 protein with the HAC followed by HAC loss from the cells as result of doxycycline removal. (b) Bright and fluorescent images of one representative clone with the HAC carrying the tTAVP64-DsRed2 cassette cultured in dox+ medium and after removal doxycycline followed by 14 days of culture. Cells were cultured without HAC selection (in HAT− and BS− medium). (c) Representative images of the metaphase spread after 14 days of culture in the presence and absence of doxycycline. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (red).
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Related In: Results  -  Collection

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

Figure 2: (a) A scheme representing binding of tTAVP64 protein with the HAC followed by HAC loss from the cells as result of doxycycline removal. (b) Bright and fluorescent images of one representative clone with the HAC carrying the tTAVP64-DsRed2 cassette cultured in dox+ medium and after removal doxycycline followed by 14 days of culture. Cells were cultured without HAC selection (in HAT− and BS− medium). (c) Representative images of the metaphase spread after 14 days of culture in the presence and absence of doxycycline. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (red).
Mentions: In contrast, a dramatic alphoidtetO HAC loss was observed in dox− medium when the tTAVP64 cassette was inserted into the HAC. Data on FISH analysis presented as a percentage of HAC-containing metaphases in different media (dox+ versus dox−) are summarized in Table 1. As seen, the absence or presence of doxycycline had essentially little, if any, effect on stability of the HAC after 3 days of culture. However, after 14 days, expression of the tTAVP64 fusion protein caused a dramatic increase in the population of cells lacking the HAC when the cells were grown in the absence of doxycycline (dox−). As a consequence, a disappearance of the DsRed2 fluorescence signal expressed from the HAC was observed when the cells were grown in the absence of doxycycline (Figure 2a and b). Note that expression of a single copy tTAVP64 from the HAC is 57-times lower than expression of the same transgene from the multiple copy vector transiently transfected into HT1080 cells (Supplementary Figure S4). Differences between two analyzed tTA cassettes is likely explained by a stronger effect of four tandem repeats of the VP16 domain in tTAVP64 on chromatin status in the HAC kinetochore compared to tTAVP16. Indeed, tTAVP64 was developed to increase the potency of transcriptional activation of tTAVP16 (36).

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