Mouse anti-Human SLC5A7 monoclonal antibody, clone 73-3F9

Background: A recently published transcript set is suitable for gene expression-based discrimination of normal colonic and colorectal cancer (CRC) biopsy samples. Our aim was to test the discriminatory power of the CRC-specific transcript set on independent biopsies and on formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: Total RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) from fresh frozen biopsies stored in RNALater (CRC (n = 15) and healthy colonic (n = 15)), furthermore from FFPE specimens including CRC (n = 15) and normal adjacent tissue (NAT) (n = 15) specimens next to the tumor. After quality and quantity measurements, gene expression analysis of a colorectal cancer-specific marker set with 11 genes (CA7, COL12A1, CXCL1, CXCL2, CHI3L1, GREM1, IL1B, IL1RN, IL8, MMP3, SLC5A7) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler (R) 480 System (Roche). In situ hybridization for two selected transcripts (CA7, CXCL1) was performed on NAT (n = 3), adenoma (n = 3) and CRC (n = 3) FFPE samples. Results: Although analytical parameters of automatically isolated RNA samples showed differences between fresh frozen biopsy and FFPE samples, both quantity and the quality enabled their application in gene expression analyses. CRC and normal fresh frozen biopsy samples could be distinguished with 93.3 % sensitivity and 86.7 % specificity and FFPE samples with 96.7 and 70.0 %, respectively. In situ hybridization could confirm the upregulation of CXCL1 and downregulation of CA7 in colorectal adenomas and tumors compared to healthy controls. Conclusion: According to our results, gene expression analysis of the analyzed colorectal cancer-specific marker set can also be performed from FFPE tissue material. With the addition of an automated workflow, this marker set may enhance the objective classification of colorectal neoplasias in the routine procedure in the future.

The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC-CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [H-3]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC-CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC-CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC-CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC-CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC-CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity. (C) 2013 Elsevier Ltd. All rights reserved.