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Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Yue Z, Carvalho A, Xu Z, Yuan X, Cardinale S, Ribeiro S, Lai F, Ogawa H, Gudmundsdottir E, Gassmann R, Morrison CG, Ruchaud S, Earnshaw WC - J. Cell Biol. (2008)

Bottom Line: However, these cells show normal sensitivity to the chemotherapeutic agent etoposide.Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth.As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

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Generation of Survivin conditional knockout in DT40 cells. (A) Diagram of the survivin locus and targeting vectors used. Arrowheads, EcoRI cleavage sites. (B) Strategy for rescue and shutoff of Survivin expression. (C) Table showing constructs used for the two knockout cell lines. (D) Southern blot of wild-type, heterozygote, and Survivin- clones. EcoRI-digested genomic DNA was hybridized with the 5′ external probe (red bar) shown in A. Superscripts 1 and 2 refer to heterozygotes from knockouts 1 and 2, respectively. (E) Repression of rescue Survivin mRNA confirmed by RT-PCR of total RNA from heterozygote and KO1 cells incubated with doxycycline. (F) Real-time PCR confirms survivin repression by doxycycline. Values were normalized relative to actin mRNA. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (G) Immunoblotting analysis of Survivin repression for KO1. 20 μg of whole cell lysate from DT40 (wild type [WT]) and SurvivinOFF cells was subjected to 12.5% SDS-PAGE and probed with affinity-purified polyclonal anti-Survivin antibody. Loading control, anti–α-tubulin. (H) Immunoblotting analysis of Survivin repression for KO2 performed as for G.
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fig1: Generation of Survivin conditional knockout in DT40 cells. (A) Diagram of the survivin locus and targeting vectors used. Arrowheads, EcoRI cleavage sites. (B) Strategy for rescue and shutoff of Survivin expression. (C) Table showing constructs used for the two knockout cell lines. (D) Southern blot of wild-type, heterozygote, and Survivin- clones. EcoRI-digested genomic DNA was hybridized with the 5′ external probe (red bar) shown in A. Superscripts 1 and 2 refer to heterozygotes from knockouts 1 and 2, respectively. (E) Repression of rescue Survivin mRNA confirmed by RT-PCR of total RNA from heterozygote and KO1 cells incubated with doxycycline. (F) Real-time PCR confirms survivin repression by doxycycline. Values were normalized relative to actin mRNA. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (G) Immunoblotting analysis of Survivin repression for KO1. 20 μg of whole cell lysate from DT40 (wild type [WT]) and SurvivinOFF cells was subjected to 12.5% SDS-PAGE and probed with affinity-purified polyclonal anti-Survivin antibody. Loading control, anti–α-tubulin. (H) Immunoblotting analysis of Survivin repression for KO2 performed as for G.

Mentions: We deleted the entire 725-bp ORF encoding survivin in chicken DT40 B lymphocytes (Fig. 1 A; Buerstedde and Takeda, 2006). Two knockouts were isolated. The first wild-type allele was replaced with a neomycin (KO1) or histidinol (KO2) selectable marker. Heterozygotes were cotransfected with two constructs, one encoding the tetracycline transactivator (tTA) plus a second with a survivin cDNA under control of a tTA-responsive promoter (tetO). The remaining allele was replaced with a histidinol (KO1) or puromycin (KO2) selectable marker (Fig. 1 C). The two knockouts differed in the control of the tTA transcription factor, which was under the control of the strong cytomegalovirus promoter in KO1 and the much weaker cellular chicken KIF4 promoter in KO2 (Fig. 1 B). Addition of doxycycline blocks tTA binding to the promoter driving the rescue cDNA, resulting in the shutoff of wild-type Survivin expression.


Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Yue Z, Carvalho A, Xu Z, Yuan X, Cardinale S, Ribeiro S, Lai F, Ogawa H, Gudmundsdottir E, Gassmann R, Morrison CG, Ruchaud S, Earnshaw WC - J. Cell Biol. (2008)

Generation of Survivin conditional knockout in DT40 cells. (A) Diagram of the survivin locus and targeting vectors used. Arrowheads, EcoRI cleavage sites. (B) Strategy for rescue and shutoff of Survivin expression. (C) Table showing constructs used for the two knockout cell lines. (D) Southern blot of wild-type, heterozygote, and Survivin- clones. EcoRI-digested genomic DNA was hybridized with the 5′ external probe (red bar) shown in A. Superscripts 1 and 2 refer to heterozygotes from knockouts 1 and 2, respectively. (E) Repression of rescue Survivin mRNA confirmed by RT-PCR of total RNA from heterozygote and KO1 cells incubated with doxycycline. (F) Real-time PCR confirms survivin repression by doxycycline. Values were normalized relative to actin mRNA. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (G) Immunoblotting analysis of Survivin repression for KO1. 20 μg of whole cell lysate from DT40 (wild type [WT]) and SurvivinOFF cells was subjected to 12.5% SDS-PAGE and probed with affinity-purified polyclonal anti-Survivin antibody. Loading control, anti–α-tubulin. (H) Immunoblotting analysis of Survivin repression for KO2 performed as for G.
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Related In: Results  -  Collection

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fig1: Generation of Survivin conditional knockout in DT40 cells. (A) Diagram of the survivin locus and targeting vectors used. Arrowheads, EcoRI cleavage sites. (B) Strategy for rescue and shutoff of Survivin expression. (C) Table showing constructs used for the two knockout cell lines. (D) Southern blot of wild-type, heterozygote, and Survivin- clones. EcoRI-digested genomic DNA was hybridized with the 5′ external probe (red bar) shown in A. Superscripts 1 and 2 refer to heterozygotes from knockouts 1 and 2, respectively. (E) Repression of rescue Survivin mRNA confirmed by RT-PCR of total RNA from heterozygote and KO1 cells incubated with doxycycline. (F) Real-time PCR confirms survivin repression by doxycycline. Values were normalized relative to actin mRNA. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (G) Immunoblotting analysis of Survivin repression for KO1. 20 μg of whole cell lysate from DT40 (wild type [WT]) and SurvivinOFF cells was subjected to 12.5% SDS-PAGE and probed with affinity-purified polyclonal anti-Survivin antibody. Loading control, anti–α-tubulin. (H) Immunoblotting analysis of Survivin repression for KO2 performed as for G.
Mentions: We deleted the entire 725-bp ORF encoding survivin in chicken DT40 B lymphocytes (Fig. 1 A; Buerstedde and Takeda, 2006). Two knockouts were isolated. The first wild-type allele was replaced with a neomycin (KO1) or histidinol (KO2) selectable marker. Heterozygotes were cotransfected with two constructs, one encoding the tetracycline transactivator (tTA) plus a second with a survivin cDNA under control of a tTA-responsive promoter (tetO). The remaining allele was replaced with a histidinol (KO1) or puromycin (KO2) selectable marker (Fig. 1 C). The two knockouts differed in the control of the tTA transcription factor, which was under the control of the strong cytomegalovirus promoter in KO1 and the much weaker cellular chicken KIF4 promoter in KO2 (Fig. 1 B). Addition of doxycycline blocks tTA binding to the promoter driving the rescue cDNA, resulting in the shutoff of wild-type Survivin expression.

Bottom Line: However, these cells show normal sensitivity to the chemotherapeutic agent etoposide.Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth.As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

Show MeSH
Related in: MedlinePlus