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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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The alphoidtetO-HAC/tTAVP64 in different cell lines. (a) Insertion of the construct tetR-VP64-IRES-DsRed2 into the loxP site of the alphoidtetO-HAC propagated in HPRT-deficient hamster CHO cells by Cre/loxP mediated recombination. (b) FISH analysis of the hamster clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. (c) From CHO cells the alphoidtetO-HAC/tTAVP64 was MMCT-transferred to human HeLa cells. (d) FISH analysis of the HeLa human clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (green).
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Figure 4: The alphoidtetO-HAC/tTAVP64 in different cell lines. (a) Insertion of the construct tetR-VP64-IRES-DsRed2 into the loxP site of the alphoidtetO-HAC propagated in HPRT-deficient hamster CHO cells by Cre/loxP mediated recombination. (b) FISH analysis of the hamster clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. (c) From CHO cells the alphoidtetO-HAC/tTAVP64 was MMCT-transferred to human HeLa cells. (d) FISH analysis of the HeLa human clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (green).

Mentions: Similar results on HAC self-elimination in dox− medium were obtained in hamster ovarian CHO cells and in human epithelial HeLa cells. For these experiments, the tetR-VP64-IRES-DsRed2 construct was inserted into a single loxP site of the alphoidtetO-HAC propagated in HPRT-minus hamster CHO cells by Cre/loxP-mediated recombination (see Materials and Methods) (Figure 4a). Several clones were selected on HAT medium. Insertion into the HAC was confirmed by PCR using specific primers (Supplementary Table S1). These primers were diagnostic for reconstitution of the HPRT gene, which accompanies Cre/loxP targeting. A FISH image of one representative clone of the HAC in CHO cells in dox+ medium after 14 days of culture is shown in Figure 4b. This FISH confirmed that the HAC is maintained autonomously. One CHO clone containing alphoidtetO-HAC/tTAVP64 was chosen for transfer to human HeLa cells via MMCT (Figure 4c). FISH analysis confirmed that the HAC propagates autonomously without detectable integration into chromosomes (Figure 4d). The results on the HAC stability in two HAC-containing clones in CHO and HeLa cell lines in dox+ versus dox− medium are summarized in Table 2. As seen, expression of the tTAVP64 cassette from the HAC induces a dramatic HAC loss in the absence of doxycycline in the analyzed cell lines after 14 days of culture (Table 2; Figure 4b and d).


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)

The alphoidtetO-HAC/tTAVP64 in different cell lines. (a) Insertion of the construct tetR-VP64-IRES-DsRed2 into the loxP site of the alphoidtetO-HAC propagated in HPRT-deficient hamster CHO cells by Cre/loxP mediated recombination. (b) FISH analysis of the hamster clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. (c) From CHO cells the alphoidtetO-HAC/tTAVP64 was MMCT-transferred to human HeLa cells. (d) FISH analysis of the HeLa human clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (green).
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Figure 4: The alphoidtetO-HAC/tTAVP64 in different cell lines. (a) Insertion of the construct tetR-VP64-IRES-DsRed2 into the loxP site of the alphoidtetO-HAC propagated in HPRT-deficient hamster CHO cells by Cre/loxP mediated recombination. (b) FISH analysis of the hamster clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. (c) From CHO cells the alphoidtetO-HAC/tTAVP64 was MMCT-transferred to human HeLa cells. (d) FISH analysis of the HeLa human clone carrying the autonomously propagated alphoidtetO-HAC/tTAVP64 in dox+ medium and after removal doxycycline followed by 14 days of culture. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a BAC32–2-mer(tetO) probe (green).
Mentions: Similar results on HAC self-elimination in dox− medium were obtained in hamster ovarian CHO cells and in human epithelial HeLa cells. For these experiments, the tetR-VP64-IRES-DsRed2 construct was inserted into a single loxP site of the alphoidtetO-HAC propagated in HPRT-minus hamster CHO cells by Cre/loxP-mediated recombination (see Materials and Methods) (Figure 4a). Several clones were selected on HAT medium. Insertion into the HAC was confirmed by PCR using specific primers (Supplementary Table S1). These primers were diagnostic for reconstitution of the HPRT gene, which accompanies Cre/loxP targeting. A FISH image of one representative clone of the HAC in CHO cells in dox+ medium after 14 days of culture is shown in Figure 4b. This FISH confirmed that the HAC is maintained autonomously. One CHO clone containing alphoidtetO-HAC/tTAVP64 was chosen for transfer to human HeLa cells via MMCT (Figure 4c). FISH analysis confirmed that the HAC propagates autonomously without detectable integration into chromosomes (Figure 4d). The results on the HAC stability in two HAC-containing clones in CHO and HeLa cell lines in dox+ versus dox− medium are summarized in Table 2. As seen, expression of the tTAVP64 cassette from the HAC induces a dramatic HAC loss in the absence of doxycycline in the analyzed cell lines after 14 days of culture (Table 2; Figure 4b and d).

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