TY - JOUR

T1 - Generation and characterization of monoclonal antibodies against the transcription factor Nkx6.1

AU - Pedersen, Inger

AU - Klinck, Rasmus

AU - Hecksher-Sorensen, Jacob

AU - Zahn, Stefan

AU - Madsen, Ole D

AU - Serup, Palle

AU - Jørgensen, Mette Christine

PY - 2006/5

Y1 - 2006/5

N2 - We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine beta-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing beta-cells, making this transcription factor a unique beta-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/kappa subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the beta-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies.

AB - We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine beta-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing beta-cells, making this transcription factor a unique beta-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/kappa subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the beta-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies.

KW - Animals

KW - Antibodies, Monoclonal

KW - Blotting, Western

KW - Embryo, Mammalian

KW - Enzyme-Linked Immunosorbent Assay

KW - Homeodomain Proteins

KW - Immunohistochemistry

KW - Mice

KW - Pancreas

KW - Rabbits

KW - Transcription Factors

U2 - 10.1369/jhc.5A6827.2006

DO - 10.1369/jhc.5A6827.2006

M3 - Journal article

C2 - 16401696

VL - 54

SP - 567

EP - 574

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 5

ER -