A humanized mouse model for the reduced folate carrier

Abstract

The ubiquitously expressed reduced folate carrier (RFC) or SLC19A1 is recognized to be an essential transport system for folates in mammalian cells and tissues. In addition to its generalized role as a folate transporter, RFC provides specialized tissue functions including absorption across intestinal/ colonic epithelia, transport across the basolateral membrane of renal proximal tubules, transplacental transport of folates, and folate transport across the blood-brain barrier. The human RFC (hRFC) gene is regulated by 5 major upstream non-coding regions (designated A1/A2, A, B, C, and D), each transcribed from a unique promoter. Altogether, at least 14 distinct hRFC transcripts can be envisaged in which different 5' untranslated regions (UTRs) are fused to a common splice acceptor region (positions -1 to -49) within the first coding exon with a common 1776 bp coding sequence. The 5' non-coding regions are characterized by alternate transcription start sites, multiple splice forms, and selective tissue distributions. Alternate 5'UTRs impact mRNA stabilities and translation efficiencies, and result in synthesis of modified hRFC proteins translated from upstream AUGs. In this report, we describe production and characterization of transgenic mice (TghRFC1) containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in both the TghRFC1 and humanized hRFC mice by real-time RT-PCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues. For the humanized hRFC mice, levels of B and A1/A2 5'UTRs predominated in all mice/tissues, thus resembling results in normal human tissues. Lower levels of A and C 5'UTRs were also detected. The availability of humanized mouse models for hRFC will permit investigators to address critical unanswered questions pertinent to human health and disease. These include the ability to analyze the hRFC gene in vivo, to control dietary and other environmental conditions that may impact levels of gene expression, and to control the genetics of the mice in order to assess the effects of hRFC gene alterations on tissue folate uptake and distribution, none of which can be easily achieved in human populations.

Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues

“…While there have been extensive studies of high frequency sequence polymorphisms in the hRFC coding and noncoding regions in relation to a variety of pathological states (4,56), these associations remain controversial. The recent production of a “humanized” RFC mouse in which the mouse RFC gene is functionally replaced by the hRFC gene locus (198) holds promise for extending in vitro findings of hRFC regulation and function including polymorphic hRFC gene variants to a clinically relevant in vivo model. This will allow correlations with dietary folate status and biological manifestations of folate deficiency.…”

Molecular genetic analysis of Down syndrome

“…This question can be restated on a molecular level as: are human genes expressed in mice regulated as if they are mouse genes or as if they are human genes. Evidence from some experiments with individual genes, for example, CBS and RFC, suggests that in terms of tissue speciWcity at least, human genes introduced into transgenic mice using large genomic DNA inserts are regulated diVerently from the cognate mouse genes and show tissue speciWcity similar to that seen in humans (Butler et al 2006;Patterson et al 2008). In an attempt to address this question more globally, Wilson et al (2008) studied mouse hepatocytes from Tc1 mice and relevant controls and concluded that genetic sequences on HSA21 largely determined the expression patterns of 121 HSA21 genes, with interspecies diVerences between mouse and human playing a secondary role.…”

SLC19A1 pharmacogenomics summary

“…A humanized mouse model for the reduced-function folate carrier has been created [15]. Expressing human SLC19A1 in transport-deficient Chinese hamster ovary cells results in restoration of MTX transport and MTX sensitivity [4].…”