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Article ; Online: Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk.

Englund, Johanna I / Ritchie, Alexandra / Blaas, Leander / Cojoc, Hanne / Pentinmikko, Nalle / Döhla, Julia / Iqbal, Sharif / Patarroyo, Manuel / Katajisto, Pekka

Development (Cambridge, England)

2021 Volume 148, Issue 12

Abstract: Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is ...

Abstract	Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.
MeSH term(s)	Animals ; Biomarkers ; Cell Differentiation/genetics ; Epithelial Cells ; Epithelium/metabolism ; Female ; Fluorescent Antibody Technique ; Gene Expression Regulation, Developmental ; Immunohistochemistry ; Laminin/genetics ; Laminin/metabolism ; Mammary Glands, Animal/embryology ; Mammary Glands, Animal/metabolism ; Mice ; Models, Biological ; Morphogenesis/genetics ; Organogenesis/genetics ; Signal Transduction ; Wnt4 Protein/genetics ; Wnt4 Protein/metabolism
Chemical Substances	Biomarkers ; Laminin ; Wnt4 Protein ; Wnt4 protein, mouse ; laminin alpha5
Language	English
Publishing date	2021-06-15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.199281
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Article ; Online: Bacterial artificial chromosomes improve recombinant protein production in mammalian cells

Bauer Anton / Eferl Robert / Musteanu Monica / Blaas Leander / Casanova Emilio

BMC Biotechnology, Vol 9, Iss 1, p

2009 Volume 3

Abstract: Abstract Background The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome ... ...

Abstract	Abstract Background The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production. Results In this work, we explore the efficacy of a Bacterial Artificial Chromosome based vector applied to production of the constant region of the human IgG1. Direct comparison of bulk HEK 293 cell cultures generated with a "conventional" vector or with a BAC-based vector showed that the BAC-based vector improved the protein yield by a factor of 10. Further analysis of stable cell clones harboring the BAC-based vector showed that the protein production was directly proportional to the number of integrated BAC copies and that the protein production was stable for at least 30 passages. Conclusion Generation of stable cell clones for protein production using Bacterial Artificial Chromosomes offers a clear advantage over the use of conventional vectors. First, protein production is increased by a factor of 10; second, protein production is stable overtime and third, generation of BAC-based expression vectors does not imply a significant amount of work compare to a conventional vector. Therefore, BAC-based vectors may become an attractive tool for protein production.
Keywords	Biotechnology ; TP248.13-248.65 ; Chemical technology ; TP1-1185 ; Technology ; T ; DOAJ:Biotechnology ; DOAJ:Life Sciences ; DOAJ:Biology and Life Sciences
Subject code	500
Language	English
Publishing date	2009-01-01T00:00:00Z
Publisher	BioMed Central
Document type	Article ; Online
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Article: A Mouse Model to Assess STAT3 and STAT5A/B Combined Inhibition in Health and Disease Conditions.

Moll, Herwig P / Mohrherr, Julian / Blaas, Leander / Musteanu, Monica / Stiedl, Patricia / Grabner, Beatrice / Zboray, Katalin / König, Margit / Stoiber, Dagmar / Rülicke, Thomas / Strehl, Sabine / Eferl, Robert / Casanova, Emilio

Cancers

2019 Volume 11, Issue 9

Abstract: Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. ...

Abstract	Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors.
Language	English
Publishing date	2019-08-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers11091226
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Article ; Online: The use of bacterial artificial chromosomes for recombinant protein production in mammalian cell lines.

Blaas, Leander / Musteanu, Monica / Grabner, Beatrice / Eferl, Robert / Bauer, Anton / Casanova, Emilio

Methods in molecular biology (Clifton, N.J.)

2012 Volume 824, Page(s) 581–593

Abstract: The choice of an expression vector is a critical step in the field of recombinant protein production in mammalian cells lines. Most expression vectors used in the field are sensitive to the surrounding chromatin to their integration site into the host ... ...

Abstract	The choice of an expression vector is a critical step in the field of recombinant protein production in mammalian cells lines. Most expression vectors used in the field are sensitive to the surrounding chromatin to their integration site into the host genome cell. This so-called chromatin positional effects influences the expression levels of the transgene and tends to silence its expression over time. Bacterial artificial chromosomes (BACs) are vectors that can accommodate inserts of up to 400 kb. Due to the large cloning capacity, BACs can harbour an entire locus with all or most of the regulatory elements controlling the expression of a gene. Therefore, BACs contain their own natural chromatin domain and are subjected to chromatin positional effects to a lesser extend or not at all. This makes cell lines generated with BAC-based expression vectors more predictable in terms of protein production and stability. In this chapter, we explore the use of BACs as expression vectors for recombinant protein production in mammalian cells.
MeSH term(s)	Animals ; Biotechnology/methods ; Blotting, Southern ; Cell Line ; Chromatin/genetics ; Chromosomes, Artificial, Bacterial/genetics ; Escherichia coli ; Genetic Vectors/genetics ; HEK293 Cells ; Homologous Recombination/genetics ; Humans ; Mice ; Polymerase Chain Reaction ; Proteins/genetics ; RNA, Untranslated ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/metabolism
Chemical Substances	Chromatin ; Gt(ROSA)26Sor non-coding RNA, mouse ; Proteins ; RNA, Untranslated ; Recombinant Proteins
Language	English
Publishing date	2012
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-433-9_31
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Article ; Online: Bacterial artificial chromosomes improve recombinant protein production in mammalian cells.

Blaas, Leander / Musteanu, Monica / Eferl, Robert / Bauer, Anton / Casanova, Emilio

BMC biotechnology

2009 Volume 9, Page(s) 3

Abstract: Background: The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. ... ...

Abstract	Background: The development of appropriate expression vectors for large scale protein production constitutes a critical step in recombinant protein production. The use of conventional expression vectors to obtain cell lines is a cumbersome procedure. Often, stable cell lines produce low protein yields and production is not stable over the time. These problems are due to silencing of randomly integrated expression vectors by the surrounding chromatin. To overcome these chromatin effects, we have employed a Bacterial Artificial Chromosome (BAC) as expression vector to obtain stable cell lines suitable for protein production. Results: In this work, we explore the efficacy of a Bacterial Artificial Chromosome based vector applied to production of the constant region of the human IgG1. Direct comparison of bulk HEK 293 cell cultures generated with a "conventional" vector or with a BAC-based vector showed that the BAC-based vector improved the protein yield by a factor of 10. Further analysis of stable cell clones harboring the BAC-based vector showed that the protein production was directly proportional to the number of integrated BAC copies and that the protein production was stable for at least 30 passages. Conclusion: Generation of stable cell clones for protein production using Bacterial Artificial Chromosomes offers a clear advantage over the use of conventional vectors. First, protein production is increased by a factor of 10; second, protein production is stable overtime and third, generation of BAC-based expression vectors does not imply a significant amount of work compare to a conventional vector. Therefore, BAC-based vectors may become an attractive tool for protein production.
MeSH term(s)	Cell Line ; Chromosomes, Artificial, Bacterial ; Genetic Engineering/methods ; Genetic Vectors ; Humans ; Immunoglobulin G/biosynthesis ; Recombinant Proteins/biosynthesis
Chemical Substances	Immunoglobulin G ; Recombinant Proteins
Language	English
Publishing date	2009-01-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1472-6750
ISSN (online)	1472-6750
DOI	10.1186/1472-6750-9-3
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Article ; Online: Mice deficient of

Dave, Kashyap / Sur, Inderpreet / Yan, Jian / Zhang, Jilin / Kaasinen, Eevi / Zhong, Fan / Blaas, Leander / Li, Xiaoze / Kharazi, Shabnam / Gustafsson, Charlotte / De Paepe, Ayla / Månsson, Robert / Taipale, Jussi

eLife

2017 Volume 6

Abstract: The gene desert upstream of ... ...

Abstract	The gene desert upstream of the
MeSH term(s)	Animals ; Carcinogenesis ; Enhancer Elements, Genetic ; Gene Expression ; Mice ; Mice, Knockout ; Proto-Oncogene Proteins c-myc/biosynthesis ; Proto-Oncogene Proteins c-myc/genetics ; Sequence Deletion
Chemical Substances	Myc protein, mouse ; Proto-Oncogene Proteins c-myc
Language	English
Publishing date	2017-06-06
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.23382
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Article ; Online: JAK-STAT signaling in hepatic fibrosis.

Mair, Markus / Blaas, Leander / Österreicher, Christoph H / Casanova, Emilio / Eferl, Robert

Frontiers in bioscience (Landmark edition)

2011 Volume 16, Issue 8, Page(s) 2794–2811

Abstract: Chronic liver injury, liver fibrosis and formation of hepatocellular carcinoma are intimately linked and represent a major medical challenge since treatment options are limited. Therefore, it is important to identify cellular and molecular pathways that ... ...

Abstract	Chronic liver injury, liver fibrosis and formation of hepatocellular carcinoma are intimately linked and represent a major medical challenge since treatment options are limited. Therefore, it is important to identify cellular and molecular pathways that promote liver damage or provide hepatoprotection for development of therapeutic approaches. Recently, the transcription factors STAT3 and STAT5 have been implicated in liver fibrosis induced by cholestatic liver damage. In this review, we summarize our current knowledge about STAT proteins in liver fibrosis and focus on common activities that underlie the hepatoprotective mechanisms regulated by IL-6/gp130/STAT3 and GH/STAT5/IGF-1 signaling pathways.
MeSH term(s)	Animals ; Carcinoma, Hepatocellular/etiology ; Carcinoma, Hepatocellular/physiopathology ; Humans ; Janus Kinases/physiology ; Liver Cirrhosis/etiology ; Liver Cirrhosis/physiopathology ; Liver Cirrhosis/prevention & control ; Liver Neoplasms/etiology ; Liver Neoplasms/physiopathology ; Liver Regeneration/physiology ; Mice ; Models, Biological ; STAT Transcription Factors/genetics ; STAT Transcription Factors/physiology ; Signal Transduction
Chemical Substances	STAT Transcription Factors ; Janus Kinases (EC 2.7.10.2)
Language	English
Publishing date	2011-06-01
Publishing country	Singapore
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2704569-9
ISSN	2768-6698 ; 1093-9946
ISSN (online)	2768-6698
ISSN	1093-9946
DOI	10.2741/3886
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Article: PhiC31-mediated cassette exchange into a bacterial artificial chromosome.

Blaas, Leander / Musteanu, Monica / Zenz, Rainer / Eferl, Robert / Casanova, Emilio

BioTechniques

2007 Volume 43, Issue 5, Page(s) 659–60, 662, 664

Abstract: The use of bacterial artificial chromosomes (BACs) modified via homologous recombination in Escherichia coli has become a powerful tool in the transgenic field. Homologous recombination allows the manipulation of BACs in very different ways. However this ...

Abstract	The use of bacterial artificial chromosomes (BACs) modified via homologous recombination in Escherichia coli has become a powerful tool in the transgenic field. Homologous recombination allows the manipulation of BACs in very different ways. However this process can be cumbersome and problematic when using large targeting constructs containing several repeated elements. In order to address this problem, we have established a phiC31 integrase-mediated cassette exchange into a BAC. As an example of this technique, we have exchanged a cassette previously recombined into a BAC containing the Rosa 26 locus, by a 16.5-kb incoming construct containing several repeated elements. The combination of homologous recombination in E. coli and cassette exchange should expand the tools for manipulating BACs, thus facilitating the generation of constructs with higher complexity.
MeSH term(s)	Bacteriophages/enzymology ; Chromosomes, Artificial, Bacterial/genetics ; Integrases/metabolism ; Mutagenesis, Insertional/methods
Chemical Substances	Integrases (EC 2.7.7.-)
Language	English
Publishing date	2007-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	48453-2
ISSN	1940-9818 ; 0736-6205
ISSN (online)	1940-9818
ISSN	0736-6205
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Article ; Online: A mouse tool for conditional mutagenesis in ovarian granulosa cells.

Grabner, Beatrice / Blaas, Leander / Musteanu, Monica / Hoffmann, Thomas / Birbach, Andreas / Eferl, Robert / Casanova, Emilio

Genesis (New York, N.Y. : 2000)

2010 Volume 48, Issue 10, Page(s) 612–617

Abstract: Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER(T)² fusion protein from a Bacterial Artificial Chromosome (BAC) containing ... ...

Abstract	Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER(T)² fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1-iCreER(T)² transgenic mice express the iCreER(T)² fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with "floxed" Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1-iCreER(T)² mice using a Cre reporter line demonstrated that Cre-mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1-iCreER(T)² mice should be a useful tool to analyze the gene functions in ovarian granulosa cells.
MeSH term(s)	Alleles ; Animals ; Chromosomes, Artificial, Bacterial/genetics ; Escherichia coli/genetics ; Female ; Genes, Reporter ; Granulosa Cells/drug effects ; Humans ; In Situ Hybridization ; Integrases/genetics ; Integrases/physiology ; Mice ; Mice, Transgenic ; Mutagenesis, Insertional/drug effects ; RNA, Messenger/metabolism ; Receptors, Estrogen/genetics ; Recombinant Fusion Proteins/biosynthesis ; Recombination, Genetic/drug effects ; Tamoxifen/pharmacology
Chemical Substances	RNA, Messenger ; Receptors, Estrogen ; Recombinant Fusion Proteins ; Tamoxifen (094ZI81Y45) ; Cre recombinase (EC 2.7.7.-) ; Integrases (EC 2.7.7.-)
Language	English
Publishing date	2010-10-01
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2004544-X
ISSN	1526-968X ; 1526-954X
ISSN (online)	1526-968X
ISSN	1526-954X
DOI	10.1002/dvg.20664
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Article ; Online: Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours.

Blaas, Leander / Pucci, Fabio / Messal, Hendrik A / Andersson, Agneta B / Josue Ruiz, E / Gerling, Marco / Douagi, Iyadh / Spencer-Dene, Bradley / Musch, Alexandra / Mitter, Richard / Bhaw, Leena / Stone, Richard / Bornhorst, Dorothee / Sesay, Abdul K / Jonkers, Jos / Stamp, Gordon / Malanchi, Ilaria / Toftgård, Rune / Behrens, Axel

Nature cell biology

2016 Volume 18, Issue 12, Page(s) 1346–1356

Abstract: The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we ... ...

Abstract	The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6
MeSH term(s)	Alleles ; Animals ; Animals, Newborn ; Breast Neoplasms/drug therapy ; Breast Neoplasms/genetics ; Breast Neoplasms/pathology ; Carcinogenesis/pathology ; Cell Lineage ; Cell Proliferation ; Clone Cells ; Disease-Free Survival ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation, Neoplastic ; Homeostasis ; Hormones/pharmacology ; Humans ; Mammary Glands, Animal/growth & development ; Mammary Glands, Animal/pathology ; Mammary Neoplasms, Experimental/genetics ; Mammary Neoplasms, Experimental/pathology ; Mice ; Neoplastic Stem Cells/metabolism ; Neoplastic Stem Cells/pathology ; Pregnancy ; Receptors, G-Protein-Coupled/metabolism ; Stem Cells/metabolism ; Stem Cells/pathology ; Up-Regulation
Chemical Substances	Hormones ; LGR6 protein, human ; Lgr6 protein, mouse ; Receptors, G-Protein-Coupled
Language	English
Publishing date	2016-10-31
Publishing country	England
Document type	Journal Article
ZDB-ID	1474722-4
ISSN	1476-4679 ; 1465-7392
ISSN (online)	1476-4679
ISSN	1465-7392
DOI	10.1038/ncb3434
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