Cloning and Mutagenesis of IKZF3 Gene

Human AIOLOS (IKZF3) ORF clone (pcDNA3.1/C-(k)DYK-IKZF3, NM_012481) was purchased from GenScript and subcloned into the mammalian expression vector pFlag-CMV2 (Sigma-Aldrich) and pcDNA3-HA using KpnI/XbaI and EcoRI/XhoI sites, respectively. Indicated mutants for the IKZF3 were generated either by the 2 step PCR method or by the site-directed mutagenesis protocol using AccuPrime Pfx DNA Polymerase (Life technologies), followed by DpnI treatment (New England Biolabs). pCMV6-AC-Myc-DDK-Human IKZF1 (IKAROS, NM_006060) and pEGFP-C1-Sumo1 (NM_003352) used in this study were previously described (12 (link), 15 (link)). pCMV3-C-HA-human HDAC1 (NM_004964.2) and pCMV3-GFPSpark-Sumo2 (BC016775) were purchased from Sino Biological. pCMV6-Entry-IKZF2 (HELIOS, NM_016260) was purchased from Origene. pcDNA3.1/C-(k)DYK-Ubiquitin B was purchased from GenScript (UBB, NM_001281720). The detailed primer sequence information is presented in Supplemental Table 6.

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Kuehn H.S., Sakovich I.S., Niemela J.E., Gil Silva A.A., Stoddard J.L., Polyakova E.A., Esteve Sole A., Aleshkevich S.N., Uglova T.A., Belevtsev M.V., Vertelko V.R., Shman T.V., Kupchinskaya A.N., Walter J.E., Fleisher T.A., Notarangelo L.D., Peng X.P., Delmonte O.M., Sharapova S.O, & Rosenzweig S.D. (2024). Disease-associated AIOLOS variants lead to immune deficiency/dysregulation by haploinsufficiency and redefine AIOLOS functional domains. The Journal of Clinical Investigation, 134(3), e172573.

Publication 2024

pFlag-CMV2 AccuPrime Pfx DNA Polymerase DpnI treatment

Corresponding Organization :

Other organizations : Belarusian Research Center For Pediatric Oncology and Hematology, University of South Florida St. Petersburg, National Institute of Allergy and Infectious Diseases, Johns Hopkins Medicine, Johns Hopkins University

Top 5 similar protocols

Variable analysis

independent variables

IKZF3 (AIOLOS) ORF clone (pcDNA3.1/C-(k)DYK-IKZF3, NM_012481)
Indicated mutants for the IKZF3 (generated either by the 2 step PCR method or by the site-directed mutagenesis protocol)

dependent variables

Not explicitly mentioned

control variables

PFlag-CMV2 (Sigma-Aldrich)
PcDNA3-HA
PCMV6-AC-Myc-DDK-Human IKZF1 (IKAROS, NM_006060)
PEGFP-C1-Sumo1 (NM_003352)
PCMV3-C-HA-human HDAC1 (NM_004964.2)
PCMV3-GFPSpark-Sumo2 (BC016775)
PCMV6-Entry-IKZF2 (HELIOS, NM_016260)
PcDNA3.1/C-(k)DYK-Ubiquitin B (UBB, NM_001281720)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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