Pancreas-Specific Cre Driver Lines and Considerations for Their Prudent Use

Cre/LoxP has broad utility for studying the function, development, and oncogenic transformation of pancreatic cells in mice. Here we provide an overview of the Cre driver lines that are available for such studies. We discuss how variegated expression, transgene silencing, and recombination in undesired cell types have conspired to limit the performance of these lines, sometimes leading to serious experimental concerns. We also discuss preferred strategies for achieving high-fidelity driver lines and remind investigators of the continuing need for caution when interpreting results obtained from any Cre/LoxP-based experiment performed in mice

Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site

BACKGROUND: Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci. RESULTS: To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection. CONCLUSIONS: The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome

Mutagenesis of diploid mammalian genes by gene entrapment

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10(−5) to 1.2 × 10(−4) per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells