Trisomy 8: a common finding in mouse embryonic stem (ES) cell lines

Background Obtaining a germ cell line is one of the most important steps in developing a transgenic or knockout mouse with a targeted mutated gene of interest. A common problem with this technology is that embryonic stem (ES) cells often lack, or are extremely inefficient at, germ line transmission. Results To determine whether chromosomal anomalies are correlated with inefficient ES cell germ line transmission, we examined 97 constructed ES cell lines using conventional cytogenetic analysis, and fluorescence in situ hybridization (FISH). Chromosomal abnormalities occurred in 44 (45%) out of the 97 specimens analyzed: 31 specimens had trisomy 8 or mosaic trisomy 8, eight specimens had partial trisomy 8 resulting from unbalanced translocations, and five specimens had other chromosomal anomalies. Conclusions Our data suggest that chromosomal analysis is an important tool for improving the yield and quality of gene targeting experiments.


Background
Although the whole human genome has now been sequenced, determining the function of each gene in the human body remains a challenge. A practical and frequent approach to studying human gene function is to use mouse models, accomplished by direct mutagenesis through targeting mouse embryonic stem (ES) cells. Established mouse ES cell lines have the ability to maintain unlimited proliferation in vitro and differentiate into a variety of cell lineages including germ cells [1]. The ability to obtain a germ line is one of the most important steps in developing a transgenic or knockout mouse with a specific mutated gene. However, a common problem with this valuable technique is that the ES cells often lack or have a low efficiency of germ line transmission.
Thus, understanding what affects the efficiency of germ line transmission is crucial to developing transgenic and knockout mice.
Many factors determine the efficiency of germ line transmission [2]. Chromosome make-up clearly affects both somatic cell chimerism and germ line transmission.
For example, ES cells with trisomy 8 are significantly less efficient at achieving other germ line transmission than cells with normal karyotypes [3]. Aneuploid ES cells have very low germ line transmission [4]. Chimeric mice obtained from chromosomally abnormal ES cells often have phenotypic abnormalities beyond those of pretargeted gene [5]. This abnormal genotype makes correlating the genotype and phenotype in chimeric mice extremely difficult, if not impossible.
It is not clear why structural and numerical chromosome abnormalities are found in ES cell lines subjected to extended culture in vitro. One hypothesis is that the changes confer a proliferative benefit to those cells (favorable selection). Cell aging is another possible explanation. The frequency of chromosomal anomalies increases with passage of cells in culture [4,6]; for example, a de novo Robertsonian translocation between homologous chromosomes 11 was spontaneously induced [7].
To qualify this troublesome phenomenon, we analyzed the chromosome of 97 mouse ES cell lines using conventional cytogenetic technique and fluorescence in situ hybridization (FISH).

Results
Of the 97 ES cell lines examined, 44 (45%) had either numerical or structural changes or a combination of both (Table 1). Among these abnormal ES cell lines, 31 had numerical changes, seven cell lines had structural changes and six cell lines had both numerical and structural changes.

Discussion
Analyses of mouse ES cell lines performed in our laboratory revealed a high rate of chromosomal abnormalities. Forty-four out of 97 ES cell lines (45%) showed abnormal karyotypes. Chromosomal abnormalities associated with chromosome 8, i.e., trisomy 8 or mosaic trisomy 8 or partial trisomy 8 due to an unbalanced translocation, accounted for 89% of all abnormalities (39 out of 44). Similar findings have been reported by other investigators [3,9,10]. ES cell clones with trisomy 8 have shown to have a selective growth advantage, and while they readily produce chimeras, they do not transmit the mutation to the germ line [3]. After trisomy 8, trisomy of chromosome 11 (6 out of 44) is the second most frequent abnormality in the karyotype analysis of ES cells, as also noted before [7,10].
The mechanism for these chromosomal abnormalities is not known, despite those frequencies. Generally, cells in vitro for numerous passages acquire chromosomal changes. The proportion of euploid cells starts to decrease abruptly after passage 15, and only 20 to 30% of cells remain euploid by passage 25 [5,11]. The efficiency of germ line transmission declines as the ES cell passage  number increases in number, at least partially due to the presence of aneuploidy in the cell population [5,12]. On average, the ES cells used for this study had undergone at least fifteen to twenty passages. For this reason, we conclude that extended culture in vitro is at least one causative factor for the high frequency of chromosomal abnormalities. Although we were unable to identify all the mouse strains in our collection, we were able to positively identify 46 ES cell lines from four major mouse strains, AB2.2, CJ7, GSI-1, and J1. Among them, 15 cell lines were derived from the AB2.2 mouse strain, 24 from CJ7, 3 from GSI-1 and 4 from J1. Interestingly, we found that the cell line AB2.2 had chromosomal abnormalities with mosaic trisomy 8, [42,XY,+Y,+8/41, XY,+Y/40,XY]. Also, its sub-ES cell lines showed various karyotypic abnormalities.
Of the 11 sub-ES cell lines, two were normal, four were trisomy 8, one was mosaic trisomy 8, two were double trisomy with chromosomes 8 and 11 and one was derivative chromosome 14 resulting from an unbalanced translocation between chromosomes 8 and 14. The most interesting finding is that one of the sub-ES cell lines showed very complex numerical chromosome abnormalities, including the most common numerical abnormalities found in our study: 40, X,-Y,+8/40,X,-Y,+11/41,X,-Y,+6,+8/41,X,-Y,+6,+11/42,XY,+6, +8/42,XY,+6,+11/40, XY. We were not able to follow up on whether or not this cell line affected other ES cell lines.

Conclusions
We have performed chromosomal analysis of 97 constructed ES cell lines. Chromosomal anomalies were seen  Total in 44(45%) out of the specimens analyzed. We found trisomy 8 to be a common anomaly in the sub-ES cell lines identified from the original mouse strains. It is not uncommon that ES cells with trisomy 8 over-proliferate. This should serve as a warning for collaborative researchers attempting to maximize their limited resources by growing more and more ES cells before sharing their clones. Our findings indicate that before the injection of ES cells into blastocysts, karyotype analysis by conventional methods including FISH analysis is needed in order to ensure genome stability.

Methods
All the experiments were performed on the established cell lines obtained from mice. No ethical issues is applied to this study.

ES cell lines
Ninety  sources, with different gene constructs. We were able to examine the AB2.2, CJ7, GSI-1 and J1 ES cell lines. Some cell lines originated from one single source and were expanded in different laboratories for at least another five to ten passages (1:3 or 1:4 splits) in culture under varying conditions. Thus, the cells used for this study had all undergone at least fifteen to twenty passages.

Chromosome preparation and karyotype analysis
The standard procedures for harvesting, making slides and staining the cells were followed [8]. ES cells were arrested in metaphase by adding colcemid (final concentration of 0.02 μg/ml) to the culture medium for one hour. The cells were then washed in phosphate buffered saline (PBS). After trypsin treatment, detached cells were spun down. A hypotonic solution [0.075 M potassium chloride(KCl)] was added, and cells were incubated for 30 min at 37°C prior to fixation. Fixation with 3:1 methanol:glacial acetic acid was performed three times prior to spreading the cells on glass slides. Twenty cells were analyzed for each line and at least five cells were karyotyped with standard nomenclature [13].

Fluorescent in situ hybridization (FISH)
The whole chromosome painting probes (WCP) for chromosomes 8, 10, 14 and 17 were purchased from a commercial source (Cambio, UK) for the ES cell lines with complex chromosomal abnormalities. FISH analysis was performed according to the manufacturer's instructions.