Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels

Liu, Xuedong; Constantinescu, Stefan N.; Sun, Yin; Bogan, Jonathan; Hirsch, David J.; Weinberg, Robert A.; Lodish, Harvey F.

doi:10.1006/abio.2000.4478

Cited by 133 publications

(138 citation statements)

References 24 publications

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“…For retroviral expression, wild-type and mutated mJAK1, mJAK2, hJAK3 and hTYK2 cDNAs were subcloned into the pMX-IRES-CD2 or pMX-IRES-CD4 biscistronic retroviral vector upstream of the IRES (Liu et al, 2000). Retroviral supernatants were generated by transient transfection of the BOSC packaging cell line with biscistronic vectors encoding the gene of interest and CD2 or CD4 as markers, and used for infection, as described (Liu et al, 2000;Seubert et al, 2003). Populations of cells expressing CD2 or CD4 above a predetermined level (top 25%) were isolated by FACS sorting.…”

Section: Methodsmentioning

confidence: 99%

JAK kinases overexpression promotes in vitro cell transformation

et al. 2007

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Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokineindependent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway.

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Section: Methodsmentioning

confidence: 99%

JAK kinases overexpression promotes in vitro cell transformation

et al. 2007

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“…A truncated form of Sp1 (Sp1DZn) lacking its DNAbinding domain that consists in three zinc-fingers (amino acids 621-708) was obtained by PCR-mediated deletion. The various constructs were sequenced before cloning into the bicistronic retroviral expression vector pMX-IRES-CD2 (a kind gift from Xuedong Liu, Boulder, CO, USA) (Liu et al, 2000). The resulting constructs code for Sp1 followed by an internal ribosomal entry site and a truncated cell surface marker (CD2) to identify transduced cells.…”

Section: Retroviral Constructions Transfections and Transductionsmentioning

confidence: 99%

Overexpression of Sp1 transcription factor induces apoptosis

et al. 2006

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Transcription factor Sp1 has recently been shown to be overexpressed in a number of human cancers and its overexpression contributes to malignant transformation. Sp1 regulates the expression of a number of genes participating in multiple aspects of tumorigenesis such as angiogenesis, cell growth and apoptosis resistance. To better understand the role of increased Sp1 levels on apoptosis regulation we have used retroviruses to overexpress this protein in haematopoietic Baf-3 cells and in 3T3 fibroblasts. We have also used inducible expression systems to control ectopic Sp1 levels in different cell types. Surprisingly, Sp1 overexpression on its own induces apoptosis in all the cellular models tested. The apoptotic pathways induced by Sp1 overexpression are cell type specific. Finally, using a truncated form of Sp1, we show that Sp1-induced apoptosis requires its DNA-binding domain. Our results highlight that Sp1 levels in untransformed cells must be tightly regulated as Sp1 overexpression leads to the induction of apoptosis. Our results also suggest that cancer cells overexpressing Sp1 can avoid Sp1-induced apoptosis.

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“…7,18 Since both proteins are coded on the same RNA, GFP expression is used as an indicator of infection efficiency and ZF expression. For screening the pMX-6ZF-VP64 library for ICAM-1 regulators, infected cells showing ICAM-1 immunofluorescence up-regulation were isolated using flow cytometry.…”

Section: Selection Of Tf Zf S Libraries In Mammalian Cells For Icam-1mentioning

confidence: 99%

In Vivo Selection of Combinatorial Libraries and Designed Affinity Maturation of Polydactyl Zinc Finger Transcription Factors for ICAM-1 Provides New Insights into Gene Regulation

Magnenat

Blancafort

Barbas

2004

Journal of Molecular Biology

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Zinc finger DNA-binding domains can be combined to create new proteins of desired DNA-binding specificity. By shuffling our repertoire of modified zinc finger domains to create randomly generated polydactyl zinc finger proteins with transcriptional regulatory domains, we developed large combinatorial libraries of zinc finger transcription factors (TF ZF s). Millions of TF ZF s can then be simultaneously screened in mammalian cells. Here, we successfully isolated specific TF ZF s that significantly positively and negatively modulate the transcription of the ICAM-1 gene in primary and cancer cells, which are relevant to ICAM-1 biology and tumor development. We show that TF ZF s can work in a general and in a cell-type specific manner depending on the regulatory domain and the zinc finger protein. We show that a TF ZF that interacts directly with the ICAM-1 promoter at an overlapping NF-kB binding enhancer can overcome or synergistically cooperate with NF-kB induction of ICAM-1. For this TF ZF , rational design was used to optimize the binding of the zinc finger protein to its DNA element and the resulting TF ZF demonstrated a direct correlation between increased affinity and efficiency of target gene regulation. Thus, combining library and affinity maturation approaches generated superior TF ZF s that may find further applications in therapeutic research and in ICAM-1 biology, and also provided novel mechanistic insights into the biology of transcription factors. Transcription factor libraries provide genome-wide approaches that can be applied towards the development of TF ZF s specific for virtually any gene or desired phenotype and may lead to the discovery of new genetic functions and pathways.

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Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels

Cited by 133 publications

References 24 publications

JAK kinases overexpression promotes in vitro cell transformation

JAK kinases overexpression promotes in vitro cell transformation

Overexpression of Sp1 transcription factor induces apoptosis

In Vivo Selection of Combinatorial Libraries and Designed Affinity Maturation of Polydactyl Zinc Finger Transcription Factors for ICAM-1 Provides New Insights into Gene Regulation

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