scholarly journals Skin-specific expression of a truncated E1a oncoprotein binding to p105-Rb leads to abnormal hair follicle maturation without increased epidermal proliferation.

1993 ◽  
Vol 121 (5) ◽  
pp. 1109-1120 ◽  
Author(s):  
C Missero ◽  
C Serra ◽  
K Stenn ◽  
G P Dotto
Keyword(s):  
Transgenic Mice ◽  
Hair Follicle ◽  
Cultured Cells ◽  
Hair Follicles ◽  
Tgf Beta ◽  
Specific Expression ◽  
Adenovirus E1a ◽  
Keratin 5 ◽  
Interfollicular Epidermis

In cultured cells, mutants of the Adenovirus E1a oncoprotein which bind to a reduced set of cellular proteins, including p105-Rb, p107, and p60-cyclin A, are transformation defective but can still interfere with exogenous growth inhibitory and differentiating signals, such as those triggered by TGF-beta. We have tested the ability of one such mutant, NTdl646, to interfere with keratinocyte growth and differentiation in vivo, in the skin of transgenic mice. Keratinocyte-specific expression of the transgene was achieved by using a keratin 5 promoter. Two independent lines of transgenic mice were obtained which expressed E1a specifically in their skin and exhibited an aberrant hair coat phenotype with striking regional variations. Affected hair shafts were short and crooked and hair follicles exhibited a dystrophic or absent inner root sheath. Interfollicular epidermis was normal, but its hyperplastic response to acute treatment with TPA (12-O-tetradecanoylphorbol-13-acetate) was significantly reduced. Primary keratinocytes derived from these animals were partially resistant to the effects of TPA and TGF-beta. The rate of spontaneous or chemically induced skin tumors in the transgenic mice was not increased. Thus, expression of a transgene which interferes with known negative growth regulatory proteins causes profound disturbances of keratinocyte maturation into a highly organized structure such as the hair follicle but does not lead to increased and/or neoplastic proliferation.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Cellular promoters incorporated into the adenovirus genome: effects of viral regulatory elements on transcription rates and cell specificity of albumin and beta-globin promoters

1986 ◽  
Vol 6 (11) ◽  
pp. 3798-3806
Author(s):  
L E Babiss ◽  
J M Friedman ◽  
J E Darnell
Keyword(s):  
Cultured Cells ◽  
Regulatory Elements ◽  
Beta Globin ◽  
Specific Expression ◽  
Adenovirus E1a ◽  
Differentiated Cells ◽  
Cell Specificity ◽  
293 Cells ◽  
Specific Factors ◽  
Globin Promoter

In the accompanying paper (Friedman et al., Mol. Cell. Biol. 6:3791-3797, 1986), hepatoma-specific expression of the rat albumin promoter within the adenovirus genome was demonstrated. However, the rate of transcription was very low compared with that of the endogenous chromosomal albumin gene. Here we show that in hepatoma cells the adenovirus E1A enhancer, especially in the presence of E1A protein, greatly stimulates transcription from the albumin promoter but not the mouse beta-globin promoter. This enhancer-dependent stimulation did not occur in myeloma cells in which a virus containing a immunoglobulin promoter and enhancer did function. These experiments suggest a limited distribution in cultured differentiated cells of cell-specific transcription factors. However, either the regulation of such cell-specific factors breaks down in other cultured cells, or strictly cell-specific factors are not at play in controlling cell-specific transcription, because HeLa cells could transcribe the albumin promoter from the same start site about 10% as well as hepatomas could and 293 cells could transcribe both albumin and globin promoters.

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression

1995 ◽  
Vol 268 (2) ◽  
pp. E213-E218 ◽  
Author(s):  
J. M. Gimble ◽  
X. Hua ◽  
F. Wanker ◽  
C. Morgan ◽  
C. Robinson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Brown Adipose ◽  
Octamer Motif

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice

1996 ◽  
Vol 270 (4) ◽  
pp. C1111-C1121 ◽  
Author(s):  
J. L. Wiedenman ◽  
I. Rivera-Rivera ◽  
D. Vyas ◽  
G. Tsika ◽  
L. Gao ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Troponin C ◽  
Type I ◽  
Initial Attempt ◽  
Specific Expression ◽  
Slow Type ◽  
Transgenic Lines ◽  
Fast Twitch Muscle ◽  
Mechanical Overload

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.

scholarly journals Plakoglobin Suppresses Epithelial Proliferation and Hair Growth in Vivo

2000 ◽  
Vol 149 (2) ◽  
pp. 503-520 ◽  
Author(s):  
Emmanuelle Charpentier ◽  
Robert M. Lavker ◽  
Elizabeth Acquista ◽  
Pamela Cowin
Keyword(s):  
Hair Growth ◽  
Cultured Cells ◽  
Hair Follicles ◽  
Tumor Formation ◽  
Hair Cycle ◽  
Epithelial Proliferation ◽  
Desmosomal Cadherins ◽  
Hair Follicle Cycle

Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein β-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of β-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH2-terminally truncated form of plakoglobin (ΔN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and ΔN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.

scholarly journals Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles.

1993 ◽  
Vol 7 (2) ◽  
pp. 204-215 ◽  
Author(s):  
M Blessing ◽  
L B Nanney ◽  
L E King ◽  
C M Jones ◽  
B L Hogan
Keyword(s):  
Transgenic Mice ◽  
Hair Follicles ◽  
Tgf Beta

scholarly journals Epithelium-Mesenchyme Interactions Control the Activity of Peroxisome Proliferator-Activated Receptor β/δ during Hair Follicle Development

2005 ◽  
Vol 25 (5) ◽  
pp. 1696-1712 ◽  
Author(s):  
Nicolas Di-Poï ◽  
Chuan Young Ng ◽  
Nguan Soon Tan ◽  
Zhongzhou Yang ◽  
Brian A. Hemmings ◽  
...  
Keyword(s):  
Hair Follicle ◽  
Hair Follicles ◽  
Peroxisome Proliferator ◽  
Follicle Development ◽  
Paracrine Factor ◽  
Peroxisome Proliferator Activated Receptor ◽  
Proliferation And Apoptosis ◽  
Normal Hair ◽  
Hair Follicle Development

ABSTRACT Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARβ/δ- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARβ/δ protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARβ/δ-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARβ/δ during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

scholarly journals Establishment of an Efficient Primary Culture System for Human Hair Follicle Stem Cells Using the Rho-Associated Protein Kinase Inhibitor Y-27632

2021 ◽  
Vol 9 ◽  
Author(s):  
Lihong Wen ◽  
Yong Miao ◽  
Zhexiang Fan ◽  
Jiarui Zhang ◽  
Yixuan Guo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hair Follicle ◽  
Culture System ◽  
Hair Follicles ◽  
Human Hair Follicle ◽  
Rock Inhibitor ◽  
Hair Follicle Stem Cells ◽  
Human Fibronectin ◽  
Follicle Stem Cells

BackgroundHair follicle tissue engineering is a promising strategy for treating hair loss. Human hair follicle stem cells (hHFSCs), which play a key role in the hair cycle, have potential applications in regenerative medicine. However, previous studies did not achieve efficient hHFSC expansion in vitro using feeder cells. Therefore, there is a need to develop an efficient primary culture system for the expansion and maintenance of hHFSCs.MethodsThe hHFSCs were obtained by two-step proteolytic digestion combined with microscopy. The cell culture dishes were coated with human fibronectin and inoculated with hHFSCs. The hHFSCs were harvested using a differential enrichment procedure. The effect of Rho-associated protein kinase (ROCK) inhibitor Y-27632, supplemented in keratinocyte serum-free medium (K-SFM), on adhesion, proliferation, and stemness of hHFSCs and the underlying molecular mechanisms were evaluated.ResultsThe hHFSCs cultured in K-SFM, supplemented with Y-27632, exhibited enhanced adhesion and proliferation. Additionally, Y-27632 treatment maintained the stemness of hHFSCs and promoted the ability of hHFSCs to regenerate hair follicles in vivo. However, Y-27632-induced proliferation and stemness in hHFSCs were conditional and reversible. Furthermore, Y-27632 maintained propagation and stemness of hHFSCs through the ERK/MAPK pathway.ConclusionAn efficient short-term culture system for primary hHFSCs was successfully established using human fibronectin and the ROCK inhibitor Y-27632, which promoted the proliferation, maintained the stemness of hHFSCs and promoted the ability to regenerate hair follicles in vivo. The xenofree culturing method used in this study provided a large number of high-quality seed cells, which have applications in hair follicle tissue engineering and stem cell therapy.

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