Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics

Kitamura, Toshio; Koshino, Yuko; Shibata, Fumi; Oki, Toshihiko; Nakajima, Hitoshi; Nosaka, Tetsuya; Kumagai, Hidetoshi

doi:10.1016/j.exphem.2003.07.005

Cited by 502 publications

(486 citation statements)

References 40 publications

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“…AID-GFP variants and GFP-AID were subcloned as EcoRI-NotI fragments into the pMXs retroviral vector. Untagged human AID and Flag-AID were subcloned as BamHI-NotI fragments into pMXs-ires-GFP 59 . AID stability monitoring is explained in supplementary methods.…”

Section: Methodsmentioning

confidence: 99%

Active nuclear import and cytoplasmic retention of activation-induced deaminase

Patenaude

Orthwein

et al. 2009

Nat Struct Mol Biol

133

175

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The enzyme Activation Induced Deaminase (AID) triggers antibody diversification in B-cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, its nuclear entry requiring active import mediated by a conformational nuclear localization sequence (NLS). We also identify a determinant for AID cytoplasmic retention in its Cterminus, which hampers diffusion to the nucleus, competes with nuclear import and is critical for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.3

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Section: Methodsmentioning

confidence: 99%

Active nuclear import and cytoplasmic retention of activation-induced deaminase

Patenaude

Orthwein

et al. 2009

Nat Struct Mol Biol

133

175

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“…Retroviral gene transfer was performed as previously described (Kitamura et al, 2003). Infected cells were sorted by FACSVantage SE (BD, Franklin Lakes, NJ, USA), or were subjected to selection in the presence of 3 mg/ml puromycin.…”

Section: Cell Culturementioning

confidence: 99%

c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis

et al. 2010

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The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (À/À) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARc in tripotent MSC-like cells and overexpressing PPARc in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

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“…The obtain full-length Dock5 cDNA, BamH1-Kpn1 fragment of RIKEN clone E130320D18 (nucleotides 249 to 1913 of Dock5 mRNA) was fused to the Kpn1-Not1 fragment of IMAGE clone 30106676 (nucleotides 1914 to 6461 of Dock5 mRNA). The whole was fused to GFP and was inserted into pMXs-puro, (23) a gift from Dr Kitamura (Tokyo, Japan). Dock5 DHR2 domain (amino acids E1119 to L1667) was cloned into pEGFP (Clontech, Mountain View, CA, USA) or myc tagged in pRs426Met.…”

Section: Plasmid Dnasmentioning

confidence: 99%

The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts

Vives

Laurin

Crès

et al. 2010

Journal of Bone and Mineral Research

106

148

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Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance because actin cytoskeleton is essential to generate the sealing zone, an osteoclast-specific structure ultimately mediating bone resorption. Here we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We discovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small-molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5 -/-mice and found that they have increased trabecular bone mass with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop antiosteoporotic treatments. ß

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Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics

Cited by 502 publications

References 40 publications

Active nuclear import and cytoplasmic retention of activation-induced deaminase

Active nuclear import and cytoplasmic retention of activation-induced deaminase

c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis

The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts

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