Genetic Engineering of Chimeric Chemokine Receptors

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding mCCR3 (Accession No.: NM_009914.4) [28 (link),29 (link),30 (link)] and mouse CCR8 (mCCR8; Accession No.: NM_007720.2) [32 (link),33 (link),34 (link)] were subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Chimeric mutants mCCR8 (mCCR3p1-38), mCCR8 (mCCR3p96-111), mCCR8 (mCCR3p176-207), and mCCR8 (mCCR3p269-285) were produced with a RAP [42 (link),43 (link)] and a MAP tag [44 (link),45 (link)] at their C-terminus using a HotStar HiFidelity polymerase kit (Qiagen Inc., Hilden, Germany). Alanine (glycine) substitutions in the mCCR3 N-terminal region were conducted using QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies Inc., Santa Clara, CA, USA). PCR fragments bearing the desired mutations were inserted into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation) using an In-Fusion HD Cloning Kit (TaKaRa Bio, Inc., Shiga, Japan).

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Tateyama N., Asano T., Suzuki H., Li G., Yoshikawa T., Tanaka T., Kaneko M.K, & Kato Y. (2022). Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry. Antibodies, 11(4), 75.

Publication 2022

pCAG-Ble vector HotStar HiFidelity polymerase kit QuikChange Lightning Site-Directed Mutagenesis Kits In-Fusion HD Cloning Kit

Alanine Ccr8 Chimeric Glycine Mouse Mutations Site directed mutagenesis Vector

Corresponding Organization : Tohoku University

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Variable analysis

independent variables

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding mCCR3 (Accession No.: NM_009914.4)
Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding mouse CCR8 (mCCR8; Accession No.: NM_007720.2)
Chimeric mutants mCCR8 (mCCR3p1-38), mCCR8 (mCCR3p96-111), mCCR8 (mCCR3p176-207), and mCCR8 (mCCR3p269-285)
Alanine (glycine) substitutions in the mCCR3 N-terminal region

dependent variables

Not explicitly mentioned

control variables

PCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)
RAP and a MAP tag at the C-terminus of the chimeric mutants
HotStar HiFidelity polymerase kit (Qiagen Inc., Hilden, Germany)
QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies Inc., Santa Clara, CA, USA)
In-Fusion HD Cloning Kit (TaKaRa Bio, Inc., Shiga, Japan)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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