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1.
PLoS One ; 12(8): e0182974, 2017.
Article in English | MEDLINE | ID: mdl-28800587

ABSTRACT

Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-ß-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.


Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing , Gene Silencing , RNA, Guide, Kinetoplastida/genetics , Smad3 Protein/antagonists & inhibitors , Actins/genetics , Actins/metabolism , Adenoviridae/genetics , Adenoviridae/metabolism , Base Sequence , Bronchi/cytology , Bronchi/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibronectins/genetics , Fibronectins/metabolism , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Genome, Human , Humans , INDEL Mutation , Primary Cell Culture , Protein Transport , RNA, Guide, Kinetoplastida/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transduction, Genetic , Transforming Growth Factor beta/pharmacology
2.
Clin Exp Metastasis ; 26(7): 673-84, 2009.
Article in English | MEDLINE | ID: mdl-19466569

ABSTRACT

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2(-/-)gammac(-/-) mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2(-/-) gammac(-/-) mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation.


Subject(s)
DNA-Binding Proteins/physiology , Disease Models, Animal , Neoplasm Metastasis , Animals , Cell Line, Tumor , DNA-Binding Proteins/genetics , Female , Flow Cytometry , Killer Cells, Natural/immunology , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, SCID , Rats
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