A protocol for rapid generation of recombinant adenoviruses using the AdEasy system

Luo, Jinyong; Deng, Zhong Liang; Luο, Xingguang; Tang, Ni; Song, Wen Xin; Chen, Jin; Sharff, Katie A.; Luu, Hue H.; Haydon, Rex C.; Kinzler, Kenneth W.; Vogelstein, Bert; He, Tong Chuan

doi:10.1038/nprot.2007.135

Cited by 758 publications

(650 citation statements)

References 26 publications

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“…The cDNA coding region to human IGFBP5 was PCR amplified and subcloned into the adenoviral shuttle vector pAdTrace-TO4 (He et al, 1998;Kang et al, 2004;Luo et al, 2007a;Kang et al, 2009). The siRNA-knockdown oligo cassettes were cloned into the shuttle vector pSES system (Luo et al, 2007b;Luo et al, 2008).…”

Section: Recombinant Adenoviral Vectors Expressing Igfbp5 and Siigfbp5mentioning

confidence: 99%

“…Cloning details and oligo sequences are available upon request. Recombinant adenovirus generation was carried out using the AdEasy system, as previously described (He et al, 1998;Kang et al, 2004;Luo et al, 2007a;Kang et al, 2009). Adenoviruses were produced in HEK293 cells and amplified to obtain high titers (He et al, 1998;Kang et al, 2004;Luo et al, 2008), resulting in the adenoviruses expressing IGFBP5 (AdR-IGFBP5) and siIGFBP5 (AdR-siIGFBP5) that also expressed RFP as a marker to monitor infection efficiency.…”

Section: Recombinant Adenoviral Vectors Expressing Igfbp5 and Siigfbp5mentioning

confidence: 99%

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Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

et al. 2011

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Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

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Section: Recombinant Adenoviral Vectors Expressing Igfbp5 and Siigfbp5mentioning

confidence: 99%

Section: Recombinant Adenoviral Vectors Expressing Igfbp5 and Siigfbp5mentioning

confidence: 99%

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

et al. 2011

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“…Adenoviral generation, infection and cell culture Sox4 adenovirus was generated using the pAdTrack system (Addgene, Cambridge, MA, USA) [37]. Viruses were purified with the Adeno-X Maxi adenovirus purification system (Clontech, Mountain View, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

SOX4 cooperates with neurogenin 3 to regulate endocrine pancreas formation in mouse models

Krentz

Tan

et al. 2015

Diabetologia

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Aims/hypothesis The sex-determining region Y (SRY)-related high mobility group (HMG) box (SOX) family of transcription factors is essential for normal organismal development. Despite the longstanding knowledge that many SOX family members are expressed during pancreas development, a role for many of these factors in the establishment of insulinproducing beta cell fate remains to be determined. The aim of this study is to elucidate the role of SOX4 during beta cell development. Methods We used pancreas and endocrine progenitor mouse knockouts of Sox4 to uncover the roles of SOX4 during pancreas development. Lineage tracing and in vitro models were used to determine how SOX4 regulates beta cell formation and understand the fate of Sox4-null endocrine lineage cells. Results This study demonstrates a progenitor cellautonomous role for SOX4 in regulating the genesis of beta cells and shows that it is required at multiple stages of the process. SOX4 deletion in the multipotent pancreatic progenitors resulted in impaired endocrine progenitor cell differentiation. Deletion of SOX4 later in the Neurog3-expressing cells also caused reductions in beta cells. Lineage studies showed loss of Sox4 in endocrine progenitors resulted in a block in terminal islet cell differentiation that was attributed to reduction in the production of key beta cell specification factors. Conclusions/interpretation These results demonstrate that SOX4 is essential for normal endocrine pancreas development both concomitant with, and downstream of, the endocrine fate decision. In conclusion, these studies position Sox4 temporally in the endocrine differentiation programme and provide a new target for improving in vitro differentiation of glucoseresponsive pancreatic beta cells.

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“…AdKGF, a replication-deficient recombinant Ad carrying the human KGF gene under the control of the cytomegalovirus (CMV) promoter, and Ad green fluorescent protein (GFP), a replication-deficient recombinant Ad carrying GFP under the control of the CMV promoter, were prepared using the pADeasy I system (Luo et al, 2007). The AdKGF virus titer was 1.8 × 10 10 plaque-forming units (pfu) per milliliter.…”

Section: Adenoviral Vectormentioning

confidence: 99%

The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

Liang

Zhao

et al. 2013

Molecules and Cells

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Epidermal stem cells (ESCs) are characterized as slowcycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGFtransfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f + /CD71 -). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing.

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A protocol for rapid generation of recombinant adenoviruses using the AdEasy system

Cited by 758 publications

References 26 publications

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma

SOX4 cooperates with neurogenin 3 to regulate endocrine pancreas formation in mouse models

The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

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