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Versatile and enhanced tumour modelling in mice via somatic cell transduction.

Rodriguez E, Mannion L, D'Santos P, Griffiths M, Arends MJ, Brindle KM, Lyons SK - J. Pathol. (2014)

Bottom Line: The vectors deliver a tamoxifen-inducible and self-inactivating Cre recombinase, conditional bioluminescent and fluorescent proteins and an shRNA component.We also show that a variant vector expressing shRNA alters tumour growth dynamics and the histological grade associated with the inherited genotype.They should also be useful for inducing imageable tumours in organs other than the lung, provided that the inherited conditional genotype is sufficiently penetrant.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Imaging, CRUK Cambridge Institute, University of Cambridge, UK.

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Related in: MedlinePlus

(a) Schematic of lentiviral vector constructs: (1) LV–LS (control vector); (2) LV–indLS1; (3) LV–indLS2; and (4) LV–indLS2miR1224; CAG; robust and ubiquitous promoter; Lox66 and lox71, mutant loxP sites; Luc_Strawberry, luciferase and mStrawberry co-expression cassette; CreERT2, tamoxifen-inducible Cre recombinase; i, synthetic intron in CreERT2; miR, miR30 cassette containing p53 (1224) hairpin. (b) Outline of the transgenic strategy to induce conditional and bioluminescent tumours in GEMs following transduction with LV–indLS2 and induction with tamoxifen: loxP and loxDM, wild-type and double-mutant lox P sites, respectively. (c) Fluorescence images of culture plates showing tamoxifen inducibility of mStrawberry expression in HEK293T cells (top panels) and bioluminescence images showing tamoxifen inducibility of luciferase expression in HEK293T cells (lower panels). (d) Flow-cytometric analysis of HEK293T cells transduced with LV–indLS2, showing transition from low- to high-level expression of mStrawberry following tamoxifen induction
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fig01: (a) Schematic of lentiviral vector constructs: (1) LV–LS (control vector); (2) LV–indLS1; (3) LV–indLS2; and (4) LV–indLS2miR1224; CAG; robust and ubiquitous promoter; Lox66 and lox71, mutant loxP sites; Luc_Strawberry, luciferase and mStrawberry co-expression cassette; CreERT2, tamoxifen-inducible Cre recombinase; i, synthetic intron in CreERT2; miR, miR30 cassette containing p53 (1224) hairpin. (b) Outline of the transgenic strategy to induce conditional and bioluminescent tumours in GEMs following transduction with LV–indLS2 and induction with tamoxifen: loxP and loxDM, wild-type and double-mutant lox P sites, respectively. (c) Fluorescence images of culture plates showing tamoxifen inducibility of mStrawberry expression in HEK293T cells (top panels) and bioluminescence images showing tamoxifen inducibility of luciferase expression in HEK293T cells (lower panels). (d) Flow-cytometric analysis of HEK293T cells transduced with LV–indLS2, showing transition from low- to high-level expression of mStrawberry following tamoxifen induction

Mentions: To induce conditional and bioluminescent tumours in floxed mice, we built a lentivirus (LV–indLS1) that contained CreERT2 (tamoxifen-inducible Cre recombinase), as well as firefly luciferase and mStrawberry expression cassettes. To couple expression of the transgenic reporters with tumour initiation and to limit the total size of the vector, we employed a single CAG promoter 21, flanked by inverted mutant loxP sites 22, to drive transgene expression. As depicted in Figure 1, only CreERT2 is expressed from the CAG promoter upon cell transduction. Following the functional activation of Cre recombinase by administration of tamoxifen, the promoter inverts and locks in the opposite orientation, resulting in expression of the reporter transgenes and Cre inactivation. Should other conditional tumour-related alleles also be present in the cell, they too should be activated or deleted by Cre recombinase. Consequently, tumours initiated in this way will be amenable to in vivo visualization by BLI.


Versatile and enhanced tumour modelling in mice via somatic cell transduction.

Rodriguez E, Mannion L, D'Santos P, Griffiths M, Arends MJ, Brindle KM, Lyons SK - J. Pathol. (2014)

(a) Schematic of lentiviral vector constructs: (1) LV–LS (control vector); (2) LV–indLS1; (3) LV–indLS2; and (4) LV–indLS2miR1224; CAG; robust and ubiquitous promoter; Lox66 and lox71, mutant loxP sites; Luc_Strawberry, luciferase and mStrawberry co-expression cassette; CreERT2, tamoxifen-inducible Cre recombinase; i, synthetic intron in CreERT2; miR, miR30 cassette containing p53 (1224) hairpin. (b) Outline of the transgenic strategy to induce conditional and bioluminescent tumours in GEMs following transduction with LV–indLS2 and induction with tamoxifen: loxP and loxDM, wild-type and double-mutant lox P sites, respectively. (c) Fluorescence images of culture plates showing tamoxifen inducibility of mStrawberry expression in HEK293T cells (top panels) and bioluminescence images showing tamoxifen inducibility of luciferase expression in HEK293T cells (lower panels). (d) Flow-cytometric analysis of HEK293T cells transduced with LV–indLS2, showing transition from low- to high-level expression of mStrawberry following tamoxifen induction
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4288983&req=5

fig01: (a) Schematic of lentiviral vector constructs: (1) LV–LS (control vector); (2) LV–indLS1; (3) LV–indLS2; and (4) LV–indLS2miR1224; CAG; robust and ubiquitous promoter; Lox66 and lox71, mutant loxP sites; Luc_Strawberry, luciferase and mStrawberry co-expression cassette; CreERT2, tamoxifen-inducible Cre recombinase; i, synthetic intron in CreERT2; miR, miR30 cassette containing p53 (1224) hairpin. (b) Outline of the transgenic strategy to induce conditional and bioluminescent tumours in GEMs following transduction with LV–indLS2 and induction with tamoxifen: loxP and loxDM, wild-type and double-mutant lox P sites, respectively. (c) Fluorescence images of culture plates showing tamoxifen inducibility of mStrawberry expression in HEK293T cells (top panels) and bioluminescence images showing tamoxifen inducibility of luciferase expression in HEK293T cells (lower panels). (d) Flow-cytometric analysis of HEK293T cells transduced with LV–indLS2, showing transition from low- to high-level expression of mStrawberry following tamoxifen induction
Mentions: To induce conditional and bioluminescent tumours in floxed mice, we built a lentivirus (LV–indLS1) that contained CreERT2 (tamoxifen-inducible Cre recombinase), as well as firefly luciferase and mStrawberry expression cassettes. To couple expression of the transgenic reporters with tumour initiation and to limit the total size of the vector, we employed a single CAG promoter 21, flanked by inverted mutant loxP sites 22, to drive transgene expression. As depicted in Figure 1, only CreERT2 is expressed from the CAG promoter upon cell transduction. Following the functional activation of Cre recombinase by administration of tamoxifen, the promoter inverts and locks in the opposite orientation, resulting in expression of the reporter transgenes and Cre inactivation. Should other conditional tumour-related alleles also be present in the cell, they too should be activated or deleted by Cre recombinase. Consequently, tumours initiated in this way will be amenable to in vivo visualization by BLI.

Bottom Line: The vectors deliver a tamoxifen-inducible and self-inactivating Cre recombinase, conditional bioluminescent and fluorescent proteins and an shRNA component.We also show that a variant vector expressing shRNA alters tumour growth dynamics and the histological grade associated with the inherited genotype.They should also be useful for inducing imageable tumours in organs other than the lung, provided that the inherited conditional genotype is sufficiently penetrant.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Imaging, CRUK Cambridge Institute, University of Cambridge, UK.

Show MeSH
Related in: MedlinePlus