Abstract
An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation.
Highlights
A decade ago, the Human Genome Project published its first human DNA sequence draft, followed shortly by the full version in 2003 [1,2,3]. This project and the SNP Consortium and the International HapMap Project have provided geneticists with invaluable tools for their research on human populations [4,5]. Their activities have resulted in an exponential growth of PubMed entries related to genome-wide association studies (GWASs) plus human whole-genome sequencing (HWGS) over the past decade (Figure 1, white bars)
Additional investigation, using a set of conserved and well-characterized transcription factors responsible for hepatocyte development and function, revealed that genetic sequence rather than interspecies differences in epigenetic machinery or cellular environment is largely responsible for directing transcriptional programs [21]. These results demonstrated that human gene regulation is generally conserved in mice, strengthening the argument that humanized mouse models (HuMMs) can be a good approach for understanding the role of candidate regulatory variants in disease development
On the other hand, assuming the challenging situation in which no heterozygous phenotype exists to complement, these females will be mated with a Yfg+/2male, resulting in males for study carrying a single copy of the human retrofitted HprtYFRV-your favourite gene (YFG) and the mouse null (Yfg2/2) gene
Summary
A decade ago, the Human Genome Project published its first human DNA sequence draft, followed shortly by the full version in 2003 [1,2,3]. It raises a problem for mouse modelling when a strictly mouse-genome-based approach is used to validate human candidate regulatory variants, since the equivalent DNA sequence and/or epigenomic environment may not be present.
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