Enhancement of the rRNA transcription rate does not increase recombinant protein synthesis.

<p>a. qRT-PCR of 45S rRNA levels in HEK293T and HeLa following expression of increasing amounts of UBF (pCMV-UBF). rRNA levels were normalized to GAPDH mRNA quantities. Data are from two independent experiments. b. SEAP expression of HEK293T and HeLa cells co-transfected with pCAG-SEAP and either pCMV-UBF or pCMV-TAP-tag (control). Data are from two independent experiments. c. Polysome profile of HEK293T cells transfected with either pCMV-UBF or pCMV-TAP-tag (control). The data show one representative experiment out of two independent experiments.</p

TIP5 knockdown.

<p>a. qRT-PCR of TIP5 mRNA of NIH/3T3 cells stably expressing shRNA-TIP5-1 and TIP5-2 sequences and of HEK293T cells stably expressing miRNA-TIP5-1 and TIP5-2 sequences. Data were normalized to GAPDH mRNA levels. Data are from two independent experiments. b. Semiquantitative RT-PCR of TIP5 mRNA of stable shRNA-TIP5-1/2 NIH/3T3, miRNA-TIP5-1/2 HEK293T and miRNA-TIP5-1/2 CHO-K1 cells. As control, RT-PCR of GAPDH mRNA is shown. Data are from two independent experiments. c. TIP5-specific Western blot analysis of stable shRNA-TIP5-1/2 NIH/3T3 and miRNA-TIP5-1/2 HEK293T cells. CHO-K1 cells could not be analyzed because the anti-TIP5 antibody does not recognize hamster TIP5. To normalize protein loading, the levels of UBF were monitored using an anti-UBF antibody.</p

Depletion of TIP5 enhances ribosome synthesis.

<p>a. Relative amounts of cytoplasmic RNA/cell in stable NIH/3T3, HEK293T and CHO-K1 cells. Data represent the average of two experiments performed in triplicate. b. Polysome profile of stable HEK293T and CHO-K1 cell lines. The data show one representative experiment out of two independent experiments. c. Depletion of TIP5 enhances production of recombinant proteins. SEAP expression of stable NIH/3T3, HEK293T and CHO-K1 cell lines engineered with the constitutive SEAP expression vector pCAG-SEAP. Data are from three independent experiments. d. Luciferase expression of stable NIH/3T3 and HEK293T cell lines engineered with the constitutive luciferase expression vector pCMV-luciferase. Data are from two independent experiments. e. Relative SEAP transcript levels of stable HEK293T cells transfected with a constitutive SEAP expression vector. Values were normalized to GAPDH mRNA levels. Data are from two independent experiments.</p

TIP5 depletion enhances rRNA production.

<p>a. Depletion of TIP5 decreases CpG methylation of rDNA promoters. Diagram of a mouse, human and Chinese hamster rDNA promoter regions including the HpaII (H) sites analyzed. Black circles indicate CpG dinucleotides. Arrows represent the primers used to amplify HpaII-digested DNA. rDNA CpG methylation levels were measured in NIH/3T3, HEK293T and CHO-K1 cells stably expressing shRNA- and/or miRNA-TIP5-1/2 and control sequences. Data represent the amounts of HpaII-resistant rDNA normalized to the total rDNA calculated by amplification with primers encompassing DNA sequences lacking HpaII-sites and undigested DNA. Data are from two independent experiments. b. Depletion of TIP5 enhances rRNA synthesis. qRT-PCR-based 45S pre-rRNA levels of stable NIH/3T3 and HEK293T cell lines were normalized to GAPDH mRNA levels. Data are from two independent experiments. c. Growth curves of NIH/3T3, HEK293T and CHO-K1 cells stably expressing miRNA-TIP5 and control sequences. Data are from two independent experiments.</p

Reversibility and adjustability of macrolide-responsive SEAP expression in HT-1080 and MCF-7 transduced by transgenic AAV type 2-derived particles

<p><b>Copyright information:</b></p><p>Taken from "Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice"</p><p>http://www.biomedcentral.com/1472-6750/7/75</p><p>BMC Biotechnology 2007;7():75-75.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2211474.</p><p></p> (A) HT-1080 and (B) MCF-7 were transduced with pDF143-derived AAV particles (2000 genomic particles/cell) and six equal populations (1–6) were cultivated in media containing different antibiotic concentrations. Cells were (i) cultivated in the presence (+++) or absence (---) of EM over 9 days, (ii) cultivated in the presence (++-) or absence (--+) of EM over 6 days and then incubated in reversed EM conditions for the remaining three days or (iii) cells were cultivated in medium whose EM status was alternated every three days +EM to --EM to +EM (+-+) or from --EM to +EM to --EM (-+-) and SEAP expression was quantified. Adjustability of SEAP expression of pDF143 (1000 genomic particles/cell) (C) and pDF51/77 (500 genomic particles/cell) (D) in HT-1080 and MCF-7 cultivated for 48 h in the presence of increasing EM concentrations. SEAP expression is shown in units/liter (U/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; SEAP, human placental secreted alkaline phosphatase. Abbreviations: EM, erythromycin; SEAP, human placental secreted alkaline phosphatase

Self- and auto-regulated AAV type 2-based expression of fluorescent proteins

<p><b>Copyright information:</b></p><p>Taken from "Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice"</p><p>http://www.biomedcentral.com/1472-6750/7/75</p><p>BMC Biotechnology 2007;7():75-75.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2211474.</p><p></p> (A) Schematic representation of an auto-regulated (pDF124), a bidirectional (pDF89) and a self-regulated (pDF141) AAV type 2-based expression unit. (B) Fluorescence micrographs of human fibrosarcoma cells (HT-1080) and a human breast cancer cell line (MCF-7) transduced with pDF124-, pDF89- and pDF141-derived AAV particles (2000 genomic particles/cell) cultivated in the presence (+) and absence (-) of EM. (C) FACS-mediated quantification of EYFP in HT-1080 transduced with equal amounts of viral particles (2000 genomic particles/cell) and cultivated in the presence (+EM) and absence (-EM) of erythromycin. Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; ETR, ET1-specific operator; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; ITR, inverted terminal repeat; pA, synthetic polyadenylation signal; pA, simian virus 40 polyadenylation signal; P, erythromycin-responsive promoter; P, minimal version of the heat-shock protein 70 promoter; P, simian virus 40 promoter

Macrolide-triggered SEAP expression in mice injected with pDF143 (ITR-P-ET1-pA-P-SEAP-pA-ITR)-derived AAV type 2 particles

<p><b>Copyright information:</b></p><p>Taken from "Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice"</p><p>http://www.biomedcentral.com/1472-6750/7/75</p><p>BMC Biotechnology 2007;7():75-75.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2211474.</p><p></p> pDF143-derived AAV particles were administered intramuscularly and SEAP levels in the serum were measured at two different time points for one group in the absence of erythromycin, and for the other group with EM injections every 24 h. SEAP expression is shown in milliunits/liter (mU/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; SEAP, human placental secreted alkaline phosphatase