Chromosomal abnormalities including deletions and translocations are commonly found in cancers. Characterization of these abnormalities led to the isolation of tumor suppressor genes and oncogenes (1,2). Chromosome rearrangement can also be achieved through radiation or chemical treatment with low efficiency (3,4,5). Cre is a recombinase that catalyzes the recombination between two LoxP sites, a 34-bp DNA element (6). If two LoxP sites are integrated into the DNA in the same direction, recombination catalyzed by Cre leads to the deletion of the interval DNA between the two LoxP sites. By targeting two LoxP sites into two different locations on the same chromosome, the Cre-LoxP system has been adopted to create specific chromosome deletions (7,8). Here, we report the generation of two retroviral vectors that can be used for generation of random chromosomal deletion.

To generate vectors for random chromosome deletion, an artificial intron with LoxP sequence was introduced into the puromycin resistant gene (see the supplementary material available online at www.BioTechniques.com). We designed two retroviral vectors (Figure 1). The puromycin-resistant gene was divided into two parts: 5′ part with LoxP and 3′ part with LoxP, which were cloned into pLIB vector (Clontech, Mountain View, CA, USA) to generate LacZ/Neo-5′Pur-LoxP-TK and Hyg-LoxP-3′Pur, respectively. With the intron, the recombinant between these two vectors can generate an intact puromycinresistant gene with the correct reading frame. In addition, vector LacZ/Neo-5′Pur-LoxP-TK contained the LacZ/Neo gene conferring resistance to G418 and the thymidine kinase (TK) gene, which provided a negative selection with gancyclovir, while vector Hyg-LoxP-3′Pur possessed a hygromycin resistance gene. After the integration of the two retroviruses, the cells were infected with adenoviruses expressing Cre recombinase, which mediated the recombination between two LoxP sites. Cells with the recombination carried an intact puromycin-resistant gene that allowed for the selection by puromycin. Cells with two retroviruses integrated on the same chromosome and in the same orientation would generate a deletion due to the recombination (Figure 1). Translocation occurred when two retroviruses were integrated on two different chromosomes. The cells with translocation could be eliminated with the treatment of gancyclovir as they, unlike those with deletion, still carried the TK gene.

Figure 1. Chromosomal deletion generated by our retroviral system based on LoxP-Cre. (Click to enlarge)

Phoenix™ Eco packaging cells (5 × 10⁶/plate; Orbigen, San Diego, CA, USA) were transfected with 15 µg LacZ/Neo-5′Pur-LoxP-TK or Hyg-LoxP-3′Pur through the calcium phosphate precipitation method. Fortyeight hours after the transfection, LacZ/Neo-5′Pur-LoxP-TK viral supernatant was added to 5 × 10⁵ NIH 3T3 cells with 8 µg/mL polybrene. Hyg-LoxP-3′Pur retrovirus was used for infection on the second day. At 48 h after retroviral infection, the cells were subjected to G418 and hygromycin selection. One week after the selection, each plate was infected with 10⁸ adenovirus expressing Cre recombinase (provided by Dr. Graham from the National Institutes of Health). The following day, the cells were selected with 2 µg/mL puromycin. About 300 clones resistant to puromycin emerged from 2 × 10⁶ cells. Six clones were isolated and expanded. When the six clones were treated with gancyclovir, three clones died, indicating that these clones contained translocations. To locate the deletion or translocation, we determined the integration sites for the 5′ long terminal repeat (LTR) of LacZ/Neo-5′Pur-LoxP-TK and the 3′ LTR of Hyg-LoxP-3′Pur through inverse PCR and sequencing. After the determination of integration sites, the recombination between the two vectors integrated at the different sites was confirmed by PCR (Figure 2). We found that three of the six clones contained deletions (see Table 1 and also the supplementary material): 1.2 Mb on chromosome 4, 7.5 Mb on chromosome 5, and 19.5 Mb on chromosome 10, demonstrating that large chromosome deletions could be effectively generated with our system. The other three clones contained translocations. As expected, these three clones were sensitive to the treatment of gancyclovir. For the clone carrying translocation between chromosome 1 and 19, we detected another insertion by the Hyg-LoxP-3′Pur vector on chromosome 5, but this insertion was found not to be involved in recombination with LacZ/Neo-5′Pur-LoxP-TK.

Figure 2. Confirmation of deletion or translocation. (Click to enlarge)

Table 1. Deletions and Translocations Created by Cre-Mediated Recombination (Click to enlarge)

The mouse genome has 2.5 × 10⁹ bp DNA that is distributed on 20 chromosomes. If the deletion is 2 Mb, and any region in the mouse genome has a 95% probability of deletion, the required number of the deletions according to the statistics based on the Poisson distribution are: LN(1-0.95)/LN[1-(2 × 10⁶/2.5 × 10⁹)] = 3.74 × 10³. Therefore, only a pool of several thousand mouse cells with chromosomal deletions needs to be generated to have deletions covering the whole genome. This technique can be used to isolate those tumor suppressor genes that are haplo-insufficient. For the regular tumor suppressor genes that require the inactivation of both alleles, inactivation of the second allele can be achieved by treating the cells with a mutagen, natural occurring point mutation, an epigenetic modification, or the recently developed Sleeping Beauty transposon system (9,10). We are currently using this system to NOG8 cells, which are diploid immortal mouse mammary epithelial cells. NOG8 cells carrying random deletions are injected subcutaneously into the nude mice for the tumor formation. Deletions containing the potential tumor suppressor genes can be identified from the tumors. This strategy can also be used to create deletion in embryonic stem cells for the generation of mice carrying large chromosome deletion.