Engineering of systematic elimination of a targeted chromosome in human cells...

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Engineering of systematic elimination of a targeted chromosome in human cells

BioMed Research International  March 7 2017

Hiroshi Sato, Hiroki Kato, Haruyoshi Yamaza, Keiji Masuda, Huong Thi Nguyen Nguyen, Thanh Thi Mai Pham, Xu Han, Yuta Hirofuji, and Kazuaki Nonaka

Abstract

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosome 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remains unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes mis-segregation of chromosome 21 after expression of Cre recombinase and subsequently, enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Introduction

Aneuploidy refers to an abnormal number of chromosomes, which is the hallmark of human tumors and can drive abnormal proliferation of cancer cells. Defective chromosome segregation during meiosis results in gametes with an abnormality in chromosome number. Normal human cells are diploid and have 46 chromosomes arranged as 22 pairs of autosomes and one pair of sex chromosomes. Most aneuploidy in human embryos is fatal, resulting in death of the fetus before birth. However, embryos with some chromosomal trisomies survive to birth with congenital disease (e.g., trisomy of chromosome 21 results in Down syndrome). Furthermore, mosaic aneuploidy results in the failure of chromosome disjunction during cell division after fertilization. Trisomy syndromes are associated with various disorders, but it is difficult to clarify the genes responsible for them. This is because over 300 genes are present even on chromosome 21, which is the smallest human autosome. Moreover, abnormal phenotypes associated with trisomy are thought to be the result of gene-dosage imbalances. Trisomy leads to increased expression of genes encoded on the extra chromosome and affects gene expression on other chromosomes, which makes it more difficult to understand disease etiology

In this study, our aim was to eliminate an entire chromosome in human cells to generate normal disomic cells from trisomic cells. Previous research has indicated that XO mice lacking the Y chromosome can be created using a pair of loxP sites in an inverted orientation. When male mice carrying the Y chromosome containing inverted loxP sites are mated with females carrying a Cre gene, chromosome loss is induced by recombination which is mediated by Cre present between the loxP sites on sister chromatids during embryogenesis. Moreover, targeted chromosome elimination has been achieved in mouse embryonic stem-somatic hybrid cells using a Cre-inverted loxP system that included a cassette consisting of green fluorescent protein (GFP) and drug-resistant genes bracketed by a pair of inverted loxP sites. To adapt this system to human cells, we developed a modified cassette containing two inverted loxP sites, in which a counter selectable gene HSV-TK was added to efficiently select cells lacking the targeted chromosome. This cassette was integrated into the target site by homologous recombination using the clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9 (CRISPR/Cas9) nickase system.

Empire Genomic's Chromosome Control 21 was used in this publication.

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