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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 of the TAR-BRV- tTAVP64 vector. This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (dot red) homologous to 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators to protect a gene from silencing (dot green). The vector also contains a YAC cassette that includes a yeast selectable marker HIS3 for selection in yeast and a yeast centromere CEN6 for proper segregation in yeast cells. The TAR-BRV- tTAVP64 vector also contains a BAC cassette that includes a F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. For gene TAR cloning experiments, the TAR-BRV- tTAVP64 vector DNA should be linearized by a unique endonuclease located between the hooks to expose targeting sequences (hooks). The TAR-BRV- tTAVP64 vector also contains a 3' HPRT-loxP cassette allowing a further loading of a TAR-gene isolate into the unique loxP site of the alphoidtetO-HAC in hamster CHO cells by Cre/loxP mediated recombination, from where the HAC may be MMCT-transferred to the recipient human cells for further gene function analysis. The TAR-BRV- tTAVP64 vector also contains the tTAVP64 chromatin modifier that is constitutively expressed from the HAC. In the presence of doxycycline the tTAVP64 cannot bind to the tetO-sequences in the alphoidtetO-HAC. (b) FISH analysis of the HAC containing the TAR-BRV- tTAVP64 vector in HT1080 cells. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a vector probe (red).
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Figure 6: (a) A scheme of the TAR-BRV- tTAVP64 vector. This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (dot red) homologous to 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators to protect a gene from silencing (dot green). The vector also contains a YAC cassette that includes a yeast selectable marker HIS3 for selection in yeast and a yeast centromere CEN6 for proper segregation in yeast cells. The TAR-BRV- tTAVP64 vector also contains a BAC cassette that includes a F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. For gene TAR cloning experiments, the TAR-BRV- tTAVP64 vector DNA should be linearized by a unique endonuclease located between the hooks to expose targeting sequences (hooks). The TAR-BRV- tTAVP64 vector also contains a 3' HPRT-loxP cassette allowing a further loading of a TAR-gene isolate into the unique loxP site of the alphoidtetO-HAC in hamster CHO cells by Cre/loxP mediated recombination, from where the HAC may be MMCT-transferred to the recipient human cells for further gene function analysis. The TAR-BRV- tTAVP64 vector also contains the tTAVP64 chromatin modifier that is constitutively expressed from the HAC. In the presence of doxycycline the tTAVP64 cannot bind to the tetO-sequences in the alphoidtetO-HAC. (b) FISH analysis of the HAC containing the TAR-BRV- tTAVP64 vector in HT1080 cells. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a vector probe (red).

Mentions: The phenomenon of HAC loss caused by expression of the tTAVP64-containing chromatin modifier from the HAC can be used to develop a powerful system to verify phenotypes induced by a gene(s) loaded into the single loxP site of the alphoidtetO-HAC. This could be achieved by combining the TAR-cloning vector (43), used to selectively isolate any full-length gene from multiple human genomes, with the tTAVP64- containing modifier cassette followed by loading of this final vector along with a gene of interest into the HAC. Figure 6a shows a diagram of a newly constructed TAR-BRV- tTAVP64 cloning vector (see details in Supplementary Figure S5). This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (red dots) homologous to the 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators (green dots) (48), such as tRNA (52,53) or cHS4 (54) or γ -satellite DNA (55) to protect the captured gene from silencing. The vector also contains a YAC cassette that includes the HIS3 marker for selection in yeast and the CEN6 centromere for proper segregation in yeast cells as well as a BAC cassette that includes the F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. This allows the TAR-isolated gene to be isolated either directly from yeast (56) or moved to bacterial cells without a step of YAC retrofitting with BAC sequence (39) for further gene DNA isolation. The TAR-BRV- tTAVP64 vector also contains a 3' HPRT-loxP cassette allowing further loading of the TAR-gene isolate into the unique loxP site of the alphoidtetO-HAC gene delivery vector in hamster HPRT-deficient CHO cells by Cre/loxP mediated recombination. From there the HAC may be MMCT-transferred (37,57) to the recipient human cells for further gene function analysis (23,38,39). Importantly, because the TAR-BRV- tTAVP64 vector contains the chromatin modifier that is constitutively expressed from the HAC, integrity of the HAC kinetochore and, as a consequence, expression of a gene of interest in the cells can be regulated. In the presence of doxycycline when the tTAVP64 does not bind to the tetO-sequences in the alphoidtetO-HAC, the HAC is mitotically stable and each cell contains a product of a gene loaded into the HAC. After removal of the ligand doxycycline, the tTAVP64 binds to tetO-sequences of the HAC resulting in HAC loss along with a gene of interest.


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 of the TAR-BRV- tTAVP64 vector. This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (dot red) homologous to 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators to protect a gene from silencing (dot green). The vector also contains a YAC cassette that includes a yeast selectable marker HIS3 for selection in yeast and a yeast centromere CEN6 for proper segregation in yeast cells. The TAR-BRV- tTAVP64 vector also contains a BAC cassette that includes a F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. For gene TAR cloning experiments, the TAR-BRV- tTAVP64 vector DNA should be linearized by a unique endonuclease located between the hooks to expose targeting sequences (hooks). The TAR-BRV- tTAVP64 vector also contains a 3' HPRT-loxP cassette allowing a further loading of a TAR-gene isolate into the unique loxP site of the alphoidtetO-HAC in hamster CHO cells by Cre/loxP mediated recombination, from where the HAC may be MMCT-transferred to the recipient human cells for further gene function analysis. The TAR-BRV- tTAVP64 vector also contains the tTAVP64 chromatin modifier that is constitutively expressed from the HAC. In the presence of doxycycline the tTAVP64 cannot bind to the tetO-sequences in the alphoidtetO-HAC. (b) FISH analysis of the HAC containing the TAR-BRV- tTAVP64 vector in HT1080 cells. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a vector probe (red).
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Related In: Results  -  Collection

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Figure 6: (a) A scheme of the TAR-BRV- tTAVP64 vector. This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (dot red) homologous to 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators to protect a gene from silencing (dot green). The vector also contains a YAC cassette that includes a yeast selectable marker HIS3 for selection in yeast and a yeast centromere CEN6 for proper segregation in yeast cells. The TAR-BRV- tTAVP64 vector also contains a BAC cassette that includes a F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. For gene TAR cloning experiments, the TAR-BRV- tTAVP64 vector DNA should be linearized by a unique endonuclease located between the hooks to expose targeting sequences (hooks). The TAR-BRV- tTAVP64 vector also contains a 3' HPRT-loxP cassette allowing a further loading of a TAR-gene isolate into the unique loxP site of the alphoidtetO-HAC in hamster CHO cells by Cre/loxP mediated recombination, from where the HAC may be MMCT-transferred to the recipient human cells for further gene function analysis. The TAR-BRV- tTAVP64 vector also contains the tTAVP64 chromatin modifier that is constitutively expressed from the HAC. In the presence of doxycycline the tTAVP64 cannot bind to the tetO-sequences in the alphoidtetO-HAC. (b) FISH analysis of the HAC containing the TAR-BRV- tTAVP64 vector in HT1080 cells. Chromosomal DNA was counterstained with DAPI (blue). The HAC was visualized using a vector probe (red).
Mentions: The phenomenon of HAC loss caused by expression of the tTAVP64-containing chromatin modifier from the HAC can be used to develop a powerful system to verify phenotypes induced by a gene(s) loaded into the single loxP site of the alphoidtetO-HAC. This could be achieved by combining the TAR-cloning vector (43), used to selectively isolate any full-length gene from multiple human genomes, with the tTAVP64- containing modifier cassette followed by loading of this final vector along with a gene of interest into the HAC. Figure 6a shows a diagram of a newly constructed TAR-BRV- tTAVP64 cloning vector (see details in Supplementary Figure S5). This vector contains restriction sites for insertion of two unique targeting sequences (hooks) (red dots) homologous to the 5' and 3' ends of a gene of interest and restriction sites for insertion of chromatin insulators (green dots) (48), such as tRNA (52,53) or cHS4 (54) or γ -satellite DNA (55) to protect the captured gene from silencing. The vector also contains a YAC cassette that includes the HIS3 marker for selection in yeast and the CEN6 centromere for proper segregation in yeast cells as well as a BAC cassette that includes the F’ factor origin of replication and the chloramphenicol acetyltransferase (CmR) gene for selection in bacterial cells. This allows the TAR-isolated gene to be isolated either directly from yeast (56) or moved to bacterial cells without a step of YAC retrofitting with BAC sequence (39) for further gene DNA isolation. The TAR-BRV- tTAVP64 vector also contains a 3' HPRT-loxP cassette allowing further loading of the TAR-gene isolate into the unique loxP site of the alphoidtetO-HAC gene delivery vector in hamster HPRT-deficient CHO cells by Cre/loxP mediated recombination. From there the HAC may be MMCT-transferred (37,57) to the recipient human cells for further gene function analysis (23,38,39). Importantly, because the TAR-BRV- tTAVP64 vector contains the chromatin modifier that is constitutively expressed from the HAC, integrity of the HAC kinetochore and, as a consequence, expression of a gene of interest in the cells can be regulated. In the presence of doxycycline when the tTAVP64 does not bind to the tetO-sequences in the alphoidtetO-HAC, the HAC is mitotically stable and each cell contains a product of a gene loaded into the HAC. After removal of the ligand doxycycline, the tTAVP64 binds to tetO-sequences of the HAC resulting in HAC loss along with a gene of interest.

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