A non-mosaic humanized mouse model of Down syndrome, trisomy of a nearly complete long arm of human chromosome 21 in mouse chromosome background

Abstract

Down syndrome (DS) is a complex human condition, and animal models trisomic for human chromosome 21 (HSA21) genes or orthologs provide insights into better understanding and treating DS. However, HSA21 orthologs are distributed into three mouse chromosomes, preventing us from generating mouse models trisomy of a complete set of HSA21 orthologs. The only existing humanized mouse DS model, Tc1, carries a HSA21 with over 20% of protein coding genes (PCGs) disrupted. More importantly, due to the human centromere, Tc1 is mosaic (a mix of euploid and trisomic cells), which makes every mouse unique and compromises interpretation of results. Here, we used mouse artificial chromosome (MAC) technology to “clone” the 34 MB long arm of HSA21 (HSA21q). Through multiple steps of microcell-mediated chromosome transfer we created a new humanized DS mouse model, Tc(HSA21q;MAC)1Yakaz (“TcMAC21”). Constitutive EGFP expression from the transchromosome and fluorescent in situ hybridization validate that TcMAC21, containing a hybrid chromosome of HSA21q and mouse centromere, is not mosaic. Whole genome sequencing shows that TcMAC21 contains a nearly complete copy of HSA21q with 93% of intact PCGs, while RNA-seq and additional mRNA/protein expression analyses confirm that PCGs are transcribed and regulated. A battery of tests show that TcMAC21 recapitulates many DS phenotypes including morphological anomalies in heart, craniofacial skeleton and brain, pathologies at molecular and cellular level, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.Significance StatementIn the last 25 years, mouse models of trisomy 21 have supported research into Down syndrome, from defining the basis for developmental effects up to support for clinical trials. However, existing models have significant shortfalls, especially for preclinical studies. These deficiencies include incomplete or inappropriate representation of trisomic genes, absence of an extra chromosome, and mosaicism.Using cutting edge technologies we produced a mouse artificial chromosome containing the entire 34Mb long arm of human chromosome 21 and, with assisted reproductive technologies, established it in the germ line of mice. This trisomic mouse manifests developmental and functional features of Down syndrome, including hippocampal-based learning and memory deficits. This is the most complete model of Down syndrome produced to date.

Longitudinal neuroanatomical and behavioral analyses show phenotypic drift and variability in the Ts65Dn mouse model of Down syndrome

“…This could include the use of multiple mouse models of DS to confirm a phenotype, or iPS cells to compare molecular and cellular phenotypes. It will be important to design careful genetic management procedures and standardized protocols for analysis of the newer models arriving in the DS research space, including the MAC21 and Ts66Yah mice ( Kazuki et al, 2020 ; Birling et al, 2017 ). It will be critical to bear in mind that all phenotypes being studied in model systems are only relevant if also present in people with DS.…”

Down syndrome

“…Consequently, these rearrangements and mosaicism necessitate cautious interpretation of studies using Tc1 mice. A new model containing 92% of the protein-coding genes on the long arm of HSA21 as a mouse artificial chromosome avoids the problem of mosaicism and has many of the DS manifestations of trisomy 21 that have been observed in previous mouse models 91 .…”

Behavioral Phenotyping for Down Syndrome in Mice

“…The Tc1 mouse model has a number of gaps in the chromosome and is mosaic, and ∼20% of the protein-coding genes of the extra chromosome are disrupted (Gribble et al, 2013). A new transchromosomal model has been made that contains ∼93% of Hsa21q (Kazuki et al, 2020). Additional testing with this mouse model is necessary to see if it effectively replicates DS phenotypes.…”

“…Even so, the mouse has proven to be a useful model organism for DS because many of the protein-coding genes currently identified on Hsa21 are conserved on Mmu 10, 16, and 17 (Yu et al, 2020). While most of the research to date has been carried out with the Ts65Dn mouse (Reeves et al, 1995), models that more closely approximate the genetic basis of DS in humans have recently been developed, involving triplication of different chromosomal regions (Kazuki et al, 2020).…”