The human lactase persistence-associated SNP ?13910*T enables in vivo functional persistence of lactase promoter–reporter transgene expression

Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.     

Expression of CD80 promoter in transgenic mice

CD80 is a very potent co-stimulatory factor which is required for complete T-cell activation. Here, we use transgenic mice as a tool to map the promoter of the CD80 gene. We engineered three different CD80 promoter driven luciferase transgenes: -3084, -1073 and -215. With these transgenes, we have generated three groups of transgenic mice. Our results showed that the -3084 CD80 promoter/luciferase transgene was sufficient to confer tissue-specific expression of the CD80 gene. When the promoter sequence was deleted to -1073, the normal tissue-specific expression was lost. A brain-specific element was mapped between -1073 nt and -215 nt. This element caused up to ninefold higher expression of the CD80 promoter/luciferase in brain tissue of -1073 CD80 promoter/luciferase transgenic animals as compared to -3084 CD80 promoter/luciferase transgenic animals. In contrast to results with a cell culture system, little luciferase activity was detected in -215 CD80 promoter/luciferase transgenic animals.     

Analysis of transcriptional regulatory sequences of the N-methyl-D-aspartate receptor 2A subunit gene in cultured cortical neurons and transgenic mice

The postnatal appearance and up-regulation of the NR2A subunit of the N-methyl-d-aspartate receptor contributes to the functional heterogeneity of the receptor during development. To elucidate the molecular mechanisms that regulate the neural and developmental specific expression of NR2A, an upstream approximately 9-kb region of the gene harboring the promoter was isolated and characterized in transgenic mice and transfected cortical neurons. Transgenic mouse lines generated with luciferase reporter constructs driven by either 9 or 1 kb of upstream sequence selectively transcribe the transgene in brain, as compared with other non-neural tissues. Reporter luciferase levels in dissociated cultures made from these mice are over 100-fold greater in neuronal/glial co-cultures than in pure glial cultures. Analysis of NR2A 5'-nested deletions in transfected cultures of cortical neurons and glia indicate that while sequences residing upstream of -1079 bp augment NR2A neuronal expression, sequences between -486 and -447 bp are sufficient to maintain neuronal preference. An RE1/NRSE element is not necessary for NR2A neuron specificity. Furthermore, comparison of the 5'-deletion constructs in cortical neurons grown for 5, 8, 11, or 14 days in vitro indicate that sequences between -1253 and -1180 bp are necessary for maturational up-regulation of NR2A. Thus, different cis-acting sequences control the regional and temporal expression of NR2A, implicating distinct regulatory pathways.     

High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence.

In our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine beta-casein promoter. The expression level of the protein in milk of 7 mice ranged from 1 to 200 microg/ml; 1 to 34 microg/ml in 6 mice and 200 microg/ml in 1 mouse. With the aim of inducing higher expression of the protein, we constructed an expression cassette comprised of 10 kb of the bovine beta-casein gene promoter and the hLF genomic sequence in place of the cDNA. The hLF genomic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine beta-casein promoter. In total, 8 transgenic mice were generated from 31 mice (transgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hLF expression cassette. Mammary-specific expression of the transgene was addressed by performing Northern hybridization of the total RNAs from various tissues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native protein. The amount of the protein in milk of 5 mice ranged from 60 to 6,600 microg/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500 microg/ml. These data suggest that the genomic lactoferrin sequence represents a valuable element for the efficient expression of the protein in milk of transgenic animals.     

VIP receptor 1 (VPAC1) promoter targets the expression of a reporter gene to cerebellum and adrenal medulla in transgenic mice

Vasoactive intestinal peptide (VIP) is a neurotransmitter with neurotropic effects. VIP functions through two distinct G-protein-coupled receptor subtypes (VPAC1 and VPAC2). We have demonstrated expression of VPAC1 in pediatric nervous system tumors, including medulloblastoma arising in the cerebellum and neuroblastoma arising in the adrenal medulla. More recently, we have reported the differentiation of neuroblastoma cells by upregulation of VIP type 1 receptor suggesting a role for VPAC1 in neuronal development.To understand the molecular mechanisms regulating VPAC1 expression in both cerebellum and adrenal medulla, we have cloned the human VPAC1 gene and sequenced 2.6-kb of the 5'-flanking sequence. Expression of the luciferase reporter gene under the control of this 2.6-kb human VPAC1 promoter was induced 35-fold in a human medulloblastoma cell line (DAOY) and 36-fold in a human neuroblastoma cell line (SKNSH). Analysis of 5'-unidirectional deletion derivatives of the 2.6-kb fragment demonstrated that a 241-bp sequence immediately upstream of the VPAC1 coding region retains high activity, suggesting that it contains the core promoter region. Quantitative RT-PCR analysis demonstrated that VPAC1 is expressed in mouse cerebellar and adrenal tissues. The VPAC1 promoter also directed expression of a reporter gene in cerebellum and adrenal medulla in transgenic mice. Along with our previous findings, these results suggest that VPAC1 may play a functional role in development of both cerebellum and adrenal medulla.     

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression.

To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns.

The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice.

These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.