Recombinant adenoviruses [mCherry-Cep63-mGFP, mGFP-Cep152-mCherry, mGFP-Cep152 (∆23)-mCherry, and mGFP-Cep152 (2D)-mCherry] were generated as described previously56 (link) by transforming the pShuttle constructs (pKM7710, pKM7737, and pKM7755, respectively) into BJ5183 cells (Addgene) for homologous recombination between the pShuttle constructs and the adenoviral pAdEasy-1 vector. Lentiviruses expressing the gene of interest were generated by cotransfecting HEK293T cells with pHR′-CMVΔR8.2Δvpr, pHR′-CMV-VSV-G (protein G of vesicular stomatitis virus), and one of the pHR′.J-CMV-SV-puro-based constructs containing a respective gene. The resulting viruses were used essentially as described previously59 (link). U2OS cells infected with the indicated adenoviruses/lentiviruses were then depleted of endogenous Cep63 or Cep152 by RNAi for 48 h before being subjected to further analysis. To maintain the expression of lentivirus-encoded Cep152 WT or mutants, cells were continuously cultured under puromycin (6 μg/mL) selection during the entire period of the experiment.
Bj5183 cells
Manufactured by Addgene
Sourced in United States
BJ5183 cells are a type of bacterial strain commonly used in molecular biology research. These cells are designed for recombination-based cloning applications, such as the construction of bacterial artificial chromosomes (BACs). BJ5183 cells contain the recA and endA mutations, which enhance their ability to efficiently recombine linear DNA fragments and improve plasmid DNA quality, respectively.
Automatically generated - may contain errors
2 protocols using bj5183 cells
1
Generating Recombinant Adenoviruses and Lentiviruses
2
Recombinant Ad-GluR6c-GFP Production
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Recombinant Ad-GluR6c-green fluorescent protein constructs were produced in accordance with standard techniques (He et al., 1998). The pAd Track CMV vector is bicistronic, and expresses both green fluorescent protein and the GluR6c domain. Briefly, GluR6c (852-908 amino acids of GluR6) was generated by polymerase chain reaction of the appropriate GluR6c coding region to incorporate lanking Bgl II and Hind III sites followed by ligation into the Ad shuttle vector pAdTrack-CMV digested with Bgl II and Hind III (Promega). The resultant plasmid was linearized by digestion with restriction endonuclease Pme I (New England Biolabs, Beverly, MA), and subsequently cotransformed into Escherichia coli (Promega). BJ5183 cells (Addgene, Cambridge, MA, USA) have an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected with kanamycin, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into Ad packaging cell lines, Human Embryonic Kidney 293 cells (Addgene). Recombinant Ads were generated typically within 7 to 12 days, purified, and then tittered.
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