Retroviral Vectors for AID Expression

Retroviral vector pMXs human AID-, AIDΔE5- and AID7.3-ires-GFP, as well as mouse AID-GFP, have been described^{24 (link),26 (link),36 (link)}. Human AID fusions were assembled as BamHI-AID-EcoRI-Linker-HindIII-AID-E58A-XhoI cassettes into pTrcHisA (ThermoFisher) or pMX-ires-GFP (Cell Biolabs). The linker encoded a flexible (SGGGG)x3 peptide. Human SPT5 was PCR amplified and cloned into a gateway-compatible mCherry-LacRep destination vector^{56 (link)} (a gift from Dr. D. Durocher, University of Toronto, Canada). Mouse AID and Linker-BirA* were PCR amplified using gateway-compatible primers and cloned into appropriate donor vectors to generate AID-BirA* fusions into a homemade gateway-compatible pMX-ires-GFP bearing a gateway cassette cloned BamHI-EcoRI by using Multisite gateway technology (Invitrogen). AID variants were generated by PCR amplification with ad hoc oligonucleotides or by quick-change site-directed mutagenesis using KOD1 DNA polymerase (Toyobo Inc.). For oligonucleotide sequences, see Supplementary Table 2.

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Methot S.P., Litzler L.C., Subramani P.G., Eranki A.K., Fifield H., Patenaude A.M., Gilmore J.C., Santiago G.E., Bagci H., Côté J.F., Larijani M., Verdun R.E, & Di Noia J.M. (2018). A licensing step links AID to transcription elongation for mutagenesis in B cells. Nature Communications, 9, 1248.

Publication 2018

pTrcHisA pMX-ires-GFP Multisite gateway technology

Cell Dna polymerase Donor Ecori Human Ires Mouse Oligonucleotide Peptide Primers Retroviral Site directed mutagenesis Vectors

Corresponding Organization :

Other organizations : Montreal Clinical Research Institute, Memorial University of Newfoundland, Sylvester Comprehensive Cancer Center, University of Miami

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Variable analysis

independent variables

Retroviral vector pMXs human AID-, AIDΔE5- and AID7.3-ires-GFP
Mouse AID-GFP
Human AID fusions assembled as BamHI-AID-EcoRI-Linker-HindIII-AID-E58A-XhoI cassettes into pTrcHisA or pMX-ires-GFP
Human SPT5 cloned into a gateway-compatible mCherry-LacRep destination vector
Mouse AID and Linker-BirA* cloned into a gateway-compatible pMX-ires-GFP bearing a gateway cassette

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: Not specified
Negative controls: Not specified

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