Cell Culture and Transfections

A CHO-K1 cell line from ATCC (Manassas, VA, USA) was cultured in Ham's F12K medium supplemented with 10% fetal bovine serum (FBS). For transient SSA assays, cells were seeded in 12-well plates at 13 × 10³ cells/well 1 day prior to transfection. Transfection was carried out the following day with 400 ng DNA using the Effectene® transfection kit (Qiagen, Hilden, Germany). Equimolar amounts of target LagoZ plasmid and either the EGFP-I-SceI N terminus-tagged protein expression vector or an enhanced green fluorescent protein (EGFP) control expression plasmid were used. The next day, medium was replaced, and the cells were incubated for another 72 h before the β-galactosidase (β-gal) assay was performed. Stably transformed cell lines were established after transfection of the CHO-K1 cell line with LagoZ vectors containing repeats of 70 and 220 bp. Forty-eight hours later, the cells were trypsinized, and transferred to 10-cm plates. G418 (400 g/mL; GIBCO BRL, Gaithersburg, MD, USA) was added after 24 h. Fourteen days later, resistant clones were screened based on their ability to yield a detectable level of DSB-induced HR (typically half of the G418^R clones).

β-Gal Activity Detection

The β-gal activity was measured in cell extracts with o-nitrophenyl-β-D-galactopyrannoside (ONPG) as a substrate. CHO-K1 cell monolayers were washed once with phosphate-buffered saline (PBS). Cells were then lysed with 10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton® X-100, and protease inhibitors for 30 min on ice, and β-gal activity was assayed. Optical density was measured at 415 nm. The β-gal activity was calculated as relative units normalized for protein concentration, incubation time, and transfection efficiency.

In Situ X-Gal Staining

CHO-K1 and CHO-K1 SSA 70 and 220 cell monolayers were fixed in 0.5% glutaraldehyde in 100 mM PBS containing 1 mM MgCl₂, 4C for 10 min. After one wash with detergent solution (100 mM PBS, 1 mM MgCl₂, 0.02% Nonidet™ P-40), the cells were incubated at 37°C in 5-bromo-4-chloro-3-indoly1-β-D-galactopranoside (X-gal) staining solution [10 mM PBS, 1 mM MgCl₂, 150 mM NaCl, 33 mM K₄Fe(CN)₆, 33 mM K₃Fe(CM)₆, 0.1% X-gal] until color development. Frequency of SSA recombination is calculated as the number of stained cells/number of I-SceI-expressin cells.

Results and Discussion

In this report we describe the study of different basic parameters of DSB-induced HR between direct repeats in CHO cells. This knowledge is increasingly important due to the increased use of DSB-induced HR in different organisms and cell types in a growing number of laboratories.

Assay Description

HR events are based on the pairing of homologous sequences, with the size (and exact degree of identity) of the homology directly impacting the efficiency of the process. In order to evaluate homology requirements for DSB-induced HR of direct repeats in mammalian cells, we designed a series of reporter plasmids with duplications of various sizes ((Figure 2)A). Our assay is based on monitoring intragenic recombination of the LagoZ reporter, induced by the I-SceI meganuclease, which has been used by many laboratories to induce cleavage and recombination in living cells and organisms. LagoZ, a derivative of the bacterial LacZ gene, codes for a functional β-gal enzyme (22). The reporter plasmids contain an internal LagoZ duplication, surrounding an SceI, cleavage site. As a consequence, the LagoZ open reading frame (ORF) is inactivated, but can be restored upon ISceI,-induced tandem repeat recombination. Restoration of LagoZ can be monitored by an ONPG dosage of the β-gal activity or by X-gal in situ staining of the cells and PCR.

Figure 2.

Homology Requirement for DSB-Induced HR of Direct Repeats

To examine the homology requirement, LagoZ constructs were cotransfected into CHO cells with either an ISceI, expression vector or a control vector, and β-gal activity was measured by a standard ONPG assay ((Figure 2)B). As shown previously, I-SceI strongly stimulates tandem repeat recombination. Low levels of DSB-induced HR are observed with only 15 bp of homology and rapidly increase with the size of the duplication, up to 70 bp. Additional increases in the size of homology result in a more moderate effect in the level of recombination and finally level out by 700 bp. In situ X-gal staining of transfected cells ((Figure 2)C) revealed that DSB-induced HR occurs in 50%–60% of the transfected cells ((Table 1)). In the absence of ISceI, a low level of background recombination is observed ((Figure 2)B). X-gal staining of transfected cells showed that this level of recombination corresponds to <0.1% of the cells ((Figure 2)C and (Table 1)).