Abstract
Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 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 remain 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 missegregation 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.
Highlights
Aneuploidy refers to an abnormal number of chromosomes, which is the hallmark of human tumors and can drive abnormal proliferation of cancer cells [1]
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 drugresistant genes bracketed by a pair of inverted loxP sites [5]. To adapt this system to human cells, we developed a modified cassette containing two inverted loxP sites, in which a counter selectable gene herpes simplex virus thymidine kinase (HSV-tk) was added to efficiently select cells lacking the targeted chromosome
HeLa cells that indicated three signals of chromosome 21 by fluorescence in situ hybridization (FISH) analysis were used in our approach (Figure 1(a))
Summary
Aneuploidy refers to an abnormal number of chromosomes, which is the hallmark of human tumors and can drive abnormal proliferation of cancer cells [1]. Embryos with some chromosomal trisomies survive to birth with congenital disease (e.g., trisomy of chromosome 21 results in Down syndrome). 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. Trisomy leads to increased expression of genes encoded on the extra chromosome and affects gene expression on other chromosomes [2, 3], which makes it more difficult to understand disease etiology
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