Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis

Abstract

DNA arrays are capable of profiling the expression patterns of many genes in a single experiment. After finding a gene of interest in a DNA array, however, labor-intensive gene-targeting experiments sometimes must be performed for the in vivo analysis of the gene function. With random gene trapping, on the other hand, it is relatively easy to disrupt and retrieve hundreds of genes͞gene candidates in mouse embryonic stem (ES) cells, but one could overlook potentially important gene-disruption events if only the nucleotide sequences and not the expression patterns of the trapped DNA segments are analyzed. To combine the benefits of the above two experimental systems, we first created Ϸ900 genetrapped mouse ES cell clones and then constructed arrays of cDNAs derived from the disrupted genes. By using these arrays, we identified a novel gene predominantly expressed in the mouse brain, and the corresponding ES cell clone was used to produced mice homozygous for the disrupted allele of the gene. Detailed analysis of the knockout mice revealed that the gene trap vector completely abolished gene expression downstream of its integration site. Therefore, identification of a gene or novel gene candidate with an interesting expression pattern by using this type of DNA array immediately allows the production of knockout mice from an ES cell clone with a disrupted allele of the sequence of interest.

Post-entrapment genome engineering: First exon size does not affect the expression of fusion transcripts generated by gene entrapment

“…For example, the average gene disrupted by LNPAT1 encoded 1268 nt of exon sequence of which an average of 596 nt of exon sequence was located downstream of the provirus. These results are similar to those that have been described for the related RET poly(A) trap vector (Matsuda et al 2004), but are at odds with studies involving VICTR3 and VICTR20 in which 44% of the integration events were reported to be near the 5Ј-ends of cellular genes (Zambrowicz et al 1998). Additional experiments will be required to identify factors that may influence the expression of fusion transcripts with multiple downstream exons.…”

“…LNPAT1 (Fig. 1) consists of the gene trap elements from the previously described RET vector (Ishida and Leder 1999) (with the longer Pol II promoter sequence [Matsuda et al 2004]) placed between heterotypic recognition sites (loxP and lox5171) for the Cre DNA site-specific recombinase (Lee and Saito 1998). An RNA instability region from the human GM-CSF gene is intended to suppress the expression of unspliced transcripts, providing further selection for cells that express properly spliced fusion transcripts.…”

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

“…Briefly, for each assay, cells were washed with phosphate-buffered saline, diluted in annexin V binding buffer containing annexin V and PI, and incubated for 15 min at room temperature in the dark. The cells were analyzed by fluorescence-activated cell sorting (BD Biosciences) as described previously (23), with the acquisition of a total 10,000 events/sample to ensure adequate data.…”

CtBP-interacting BTB Zinc Finger Protein (CIBZ) Promotes Proliferation and G1/S Transition in Embryonic Stem Cells via Nanog

“…A 8.8 kb fragment and a 1.3 kb fragment, obtained by PCR, were used as the long and short arms for the targeting vector. The resulting targeting vector was linearized with XhoI digestion and introduced into RF8 ESCs by electroporation and selected with G418 (200 g/ml) according to the protocol described (16). Genomic DNAs from G418-resistant colonies were screened for homologous recombination by Southern blotting and PCR.…”

“…Cell Culture, siRNA, and Transient Transfection-Mouse RF8 ESCs were maintained on mitomycin-treated mouse embryonic fibroblasts (MEFs) in standard ESC culture medium (DMEM, 15% fetal bovine serum, 2 mM L-glutamine, 100 M nonessential amino acids, 1% penicillin and streptomycin, and 0.1 mM ␤-mercaptoethanol), as previously described (16). ESCs were transfected with 50 nM CIBZ-specific or scrambled negative control Dicer substrate siRNA duplexes (Integrated DNA Technologies), as described previously (18).…”