Development of an inducible gene expression system for primary murine keratinocytes

Nagarajan, Priyadharsini; Sinha, Satrajit

doi:10.1016/j.jdermsci.2007.09.003

Cited by 6 publications

(5 citation statements)

References 40 publications

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“…In this investigation we have studied chromosome territory organization during differentiation of human keratinocyte cells in culture following induction with calcium chloride (Boyce and Ham, 1983; Sacks et al, 1985; Poumay and Leclercq‐Smekens, 1998; Tu et al, 2004). This 2D keratinocyte cell system mimics the changes in gene expression that occur during in vivo keratinocyte differentiation (Green, 1980; Banks‐Schlegel and Green, 1981; Watt, 1983) including three distinct stages of differentiation—early, mid, and late (Eckert et al, 1997b; Micallef et al, 2008; Nagarajan and Sinha, 2008). Each of these stages involve a highly orchestrated expression of a specific subset of genes (Stanley and Yuspa, 1983; Eckert, 1989; Fuchs, 1994; Fuchs and Weber, 1994; Eckert and Welter, 1996; Eckert et al, 1997a,b, 2004).…”

Section: Discussionmentioning

confidence: 99%

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Chromosomal rearrangements during human epidermal keratinocyte differentiation

Marella

Seifert

Nagarajan

et al. 2009

Journal Cellular Physiology

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Undifferentiated human epidermal keratinocytes are self-renewing stem cells that can be induced to undergo a program of differentiation by varying the calcium chloride concentration in the culture media. We utilize this model of cell differentiation and a 3D chromosome painting technique to document significant changes in the radial arrangement, morphology, and interchromosomal associations between the gene poor chromosome 18 and the gene rich chromosome 19 territories at discrete stages during keratinocyte differentiation. We suggest that changes observed in chromosomal territorial organization provides an architectural basis for genomic function during cell differentiation and provide further support for a chromosome territory code that contributes to gene expression at the global level.

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Section: Discussionmentioning

confidence: 99%

“…Keratinocytes in the late stage were labeled with antibodies against filaggrin. All primary antibodies use in these studies have been previously described (Nagarajan and Sinha, 2008). In all cases secondary antibodies conjugated to Alexa594 (Invitrogen) were used for immunofluorescence detection.…”

Section: Methodsmentioning

confidence: 99%

Chromosomal rearrangements during human epidermal keratinocyte differentiation

Marella

Seifert

Nagarajan

et al. 2009

Journal Cellular Physiology

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“…We have grown and propagated IMOK cells in a custom-formulated low calcium medium, which is identical to media widely used to grow mouse epidermal keratinocytes (11). Under these culture conditions, the proliferative capacity and the differentiation potential of the IMOK cells do not seem to vary over a wide range of passage numbers (our unpublished data).…”

Section: Discussionmentioning

confidence: 99%

“…The most frequently cultured keratinocytes are from epidermal origin and established protocols are available to isolate and expand epidermal keratinocytes and to induce their differentiation in culture (10, 11). For example, elevated extra-cellular calcium or the addition of phorbol esters or retinoic acid are able to induce epidermal keratinocyte differentiation in culture (12–14).…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin

Parikh

Nagarajan

Matsui

et al. 2008

Archives of Oral Biology

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Objective-To establish an oral epithelial cell line of mouse origin for molecular and biochemical assays.Design-Epithelial cells were isolated from the oral cavity of adult mice and established as a spontaneously immortalized cell line in culture, designated immortalized oral keratinocyte cells (IMOK cells). The cells were then characterized for growth characteristics, differentiation potential, karyotype, transfectability, susceptibility to viral infection and responses to siRNA.Results-The IMOK cells exhibit robust growth in both serum-containing and serum-free medium for at least 100 population doublings. IMOK cells have a near diploid karyotype, express keratinocyte marker proteins and can be induced to undergo differentiation by addition of high levels of calcium to the medium. The differentiation process is characterized by morphological changes and by the induction of oral epithelium specific differentiation marker sproteins such as K4 and K13. Transient transfection analyses reveal that IMOK cells are highly transfectable and that several promoters of epithelial cells are active in these cells. Moreover, upon differentiation with calcium, there is an upregulation of differentiation-specific K4 and Elf5 promoter activity. Finally, we show that the oral keratinocytes are also amenable to infection with retroviruses and to siRNA based knock-down of gene expression.Conclusions-Our study is the first to establish an immortalized oral keratinocyte cell line of murine origin that can recapitulate the oral epithelium differentiation program and thus could serve as a useful tool for toxicological and molecular analyses of the oral tissue.

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“…The use of retroviruses, knock-in constructs and stable transfections for molecular reprogramming can lead to insertional mutagenesis over time (Goessler et al, 2006;Hanna et al, 2007). There are significant costs, equipment and time involved in creating stable cell lines, along with potential harmful effects of the continuous antibiotic selection pressure (Nagarajan and Sinha, 2008;Rosser et al, 2005). Furthermore, there is a need for validation testing of the cloned cell lines to ensure batch homogeneity and minimal phenotypic changes from the starter passage (Soliman et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Transient gene delivery for functional enrichment of differentiating embryonic stem cells

Wallenstein¹,

Barminko²,

Schloss³

et al. 2008

Biotech & Bioengineering

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There is a critical need for new sources of hepatocytes, both clinically to provide support for patients with liver failure and in drug discovery for toxicity, metabolic and pharmacokinetic screening of new drug entities. We have reported previously a variety of methods for differentiating murine embryonic stem (ES) cells into hepatocyte-like cells. One major challenge of our work and others in the field has been the ability to selectively purify and enrich these cells from a heterogeneous population. Traditional approaches for inserting new genes (e.g., stable transfection, knock-in, retroviral transduction) involve permanent alterations in the genome. These approaches can lead to mutations and involve the extra costs and time of developing, validating and maintaining new cell lines. We have developed a transient gene delivery system that uses fluorescent gene reporters for purification of the cells. Following a transient transfection, the cells are purified through a fluorescence-activated cell sorter (FACS), re-plated in secondary culture and subsequent phenotypic analysis is performed. In an effort to test the ability of the reporters to work in a transient environment for our differentiation system, we engineered two non-viral plasmid reporters, the first driven by the mouse albumin enhancer/promoter and the second by the mouse cytochrome P450 7A1 (Cyp7A1) promoter. We optimized the transfection efficiency of delivering these genes into spontaneously differentiated ES cells and sorted independent fractions positive for each reporter 17 days after inducing differentiation. We found that cells sorted based on the Cyp7A1 promoter showed significant enrichment in terms of albumin secretion, urea secretion and cytochrome P450 1A2 detoxification activity as compared to enrichment garnered by the albumin promoter-based cell sort. Development of gene reporter systems that allow us to identify, purify and assess homogeneous populations of cells is important in better understanding stem cell differentiation pathways. And engineering cellular systems without making permanent gene changes will be critical for the generation of clinically acceptable cellular material in the future.

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Development of an inducible gene expression system for primary murine keratinocytes

Cited by 6 publications

References 40 publications

Chromosomal rearrangements during human epidermal keratinocyte differentiation

Chromosomal rearrangements during human epidermal keratinocyte differentiation

Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin

Transient gene delivery for functional enrichment of differentiating embryonic stem cells

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