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.
Aneuploidy refers to an abnormal number of chromosomes, which is the hallmark of human tumors and can drive abnormal proliferation of cancer cells [
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 [
HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (4.5 g/l glucose) (DMEM: Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, MO, USA), 100 units/ml penicillin, and 100
Hela cells cultured on 10 cm dishes were harvested with 0.25% Trypsin-EDTA (Gibco, NY, USA). Cells were treated with 0.075 M KCl for 40 min, fixed with ethanol-acetic acid, 3 : 1 (v/v), and placed on glass slides. For FISH analysis of chromosome 21, hybridization was performed according to the manufacturers’ instructions using the chromosome 21 control probe labelled green 5-fluorescein dUTP (CHR21-10-GR, Empire Genomics, NY, USA) that specifically hybridizes to the centromeric region of chromosome 21. Following hybridization, slides were counterstained using 0.1
To create the donor vector, cassettes consisting of marker genes NeoR, EGFP, and HSV-tk and inverted loxP sequences were constructed into the EcoRI–BamHI site of pBluescript SK. Each of the 3 kbp sequences flanking the integration site was amplified from the genomic DNA using PCR (primers: 5′-CCCCCTCGAGGTCGAAGAAATGAGCTGTCCGGCTA-3′ and 5′-TATCGATACCGTCGACAGTTTCAGTTGGACACCGA-3′ or 5′-CAGAAGCTGGGGATCTGAAATTCTGAGGCTGTTGA-3′ and 5′-TAGAACTAGTGGATCAATCACCTTCCGTCCTTCCT-3′), and they were cloned into the SalI or BamHI site of the vector plasmid by InFusion (Takara, Kyoto, Japan).
Integration of the DNA cassette into the HeLa genome was performed using the CRISPR/Cas9 nickase system. The sgRNA sequence was designed using CRISPR Direct (
Cells were seeded on six-well plates at a concentration of 1.5 × 105 cells/well 1 day before transfection. Then, 500 ng of the Cre expression vector pCMV-Cre or pEGFP-N2 was transfected using Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. Cells were grown for 3 days, and then they were trypsinized and replated on 10 cm dishes in selection medium with 1
Genomic DNA was isolated from each HeLa cell and used as a template for PCR. PCR amplification was performed using KOD FX Neo Polymerase (Toyobo, Osaka, Japan). Primer 1 and 2 sequences were 5′-GTGCATTGTTTCAAGCCACTACGTTTATGA-3′ and 5′-GGGACTGAAGTTCCATCCAA-3′, respectively.
Cells were seeded at a density of 5 × 104 per well in a six-well plate and grown at 37°C in 5% CO2. The cells were harvested with 0.25% Trypsin-EDTA (Gibco, NY, USA) and mixed with an equal volume of 0.4% Trypan Blue solution (Wako, Osaka, Japan). Cell number and viability were determined using a hemocytometer. To calculate cell viability, at least 200 cells were counted in each culture after four days.
We targeted chromosome 21 for elimination in these experiments. HeLa cells that indicated three signals of chromosome 21 by fluorescence in situ hybridization (FISH) analysis were used in our approach (Figure
Strategy to eliminate a copy of chromosome 21 in HeLa cells. (a) Image of HeLa cell nuclei indicating three signals (green) by FISH using a chromosome 21 specific probe. DNA was stained with DAPI. (b) Scheme of recombination at inverted loxP sites by Cre recombinase. DNA cassette was integrated into chromosome 21 by homology-directed repair induced CRISPR/Cas9 nickase system. Cre-mediated recombination at the loxP sites between sister chromatids produced a dicentric and an acentric chromosome. Neo: neomycin resistance gene.
To induce recombination between loxP sites, the Cre recombinase expression vector pCMV-Cre was transfected into the HeLa cell-integrated DNA cassette. Cre recombinase can mediate two types of recombination between loxP sites on an identical chromosome or between loxP sites on replicated sister chromatids. Recombination in an identical chromosome leads to inversion of the fragment bracketed by loxP sites. On the other hand, when loxP sites between sister chromatids are recombined, an acentric chromosome fragment and a dicentric chromosome harboring two centromeres are generated (Figure
Recombination analysis between loxP sites by Cre recombinase. PCR primer-binding sites were shown on acentric chromosome and normal chromosome 21. PCR amplification was performed using genomic DNA isolated from the cells with and without integration of the DNA cassette before (Cre−) or 3 days after transfection of pCMV-Cre (Cre+).
These dicentric and acentric chromosomes are unstable during mitosis and would be lost from the daughter cells because of missegregation. Cells eliminating the targeted chromosome 21 could be selected by inclusion of the antiviral drug ganciclovir (GCV) in the medium. Cells including targeted chromosome 21 were killed in this selection medium because of HSV-tk genes present in the target fragment. Because of this selection, surviving cells formed colonies on the dish. EGFP signals were not detected in the selected cells but were observed in the cells before Cre expression (Figure
HeLa cells eliminated one copy of chromosome 21. (a) Observation of GFP expression of the cells without pCMV-Cre transfection (Cre−) or with pCMV-Cre transfection and GCV selection (Cre+, GCV+). (b) Image of the nuclei from cells selected by GCV by FISH using chromosome 21 specific probe (green). DNA was stained with DAPI. (c) Colony formation of the cells transfected with pCMV-Cre or control (pEGFP-N2). The number of colonies was estimated in the selection medium containing GCV. (d) The effect of eliminating one copy of chromosome 21 on cell growth. A HeLa cell with three copies of chromosome 21 and two clones in which a copy of chromosome 21 was eliminated, clone 1 and clone 2. These clones were isolated independently. (e) Viability of the HeLa cell and two clones. Error bars represent the standard deviation calculated from three independent experiments. Asterisks indicate significant differences (
Some surviving colonies were present after control plasmid transfection, indicating that the targeted chromosome 21 could be lost spontaneously from HeLa cells in the selection medium (Figure
To investigate the effects of the elimination of one copy of chromosome 21 in the HeLa cell, cell growth and viability were calculated (Figures
In this study, we developed the system to eliminate the targeted chromosome 21 from human cells. Besides trisomy of chromosome 21, congenital syndromes are caused by extra copies of other chromosomes, such as trisomy of chromosomes 13 or 18, and extra copies of the X or Y chromosome. In these cases, cells with normal copy numbers of chromosomes can be generated from patient cells. Therefore, this system could be useful in the etiological analysis of chromosome abnormality phenotypes using cultured cells and for the preparation of immune tolerance sources for autotransplantation therapy.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was supported by JSPS KAKENHI Grant no. JP16K15839, a grant-in-aid for Exploratory Research to K. Nonaka.