pSAT, the splice acceptor test plasmid. Therefore, it would be interesting to carry out insertional mutagenesis experiments using the RET vector and retrovirally infectable CHO-K1 cells looking for some mutant phenotype. Ashland Grease Trap 4815 is one of the best because of its high-end protection, low maintenance, and intelligent design. We have described a versatile retrovirus vector, RET, which should be useful in a wide range of gene-trap experiments. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. Two rounds of 3′ RACE was performed in a 50 µl reaction using the Advantage-GC cDNA polymerase mix (Clontech), first with 1 µl of the above cDNA mix (1/20 of the total reaction), NEO 1.5 primer (5′-GCGAATGGGCTGACCGCTTCCTCGTGC-3′) and AD primer (5′-CGTAGCTCTAGACTCCGTGTCCAAC-3′), and second with 1 µl of the first PCR mix (1/50 of the total reaction), NEO 2.0 primer (5′-TACGGTATCGCCGCTCCCGATTCGCAG-3′) and AD-plus primer (5′-CGTAGCTCTAGACTCCGTGTCCAACTTTT-3′). After infection and HAT selection of NIH 3T3 tk(−) cells, 400 HAT-resistant colonies for each virus construct were further grown in the medium containing G418 and HT. Convenient positioning: Strengthened tubing allows for trap to be positioned upright for direct monitoring of sample collection. Les poulies trapézoïdales à moyeu amovible SPB à 2 gorges (PMA) sont des poulies trapézoïdales poly-v à moyeu amovible, fabriquées en fonte. The GFP serves as a visible marker of transcriptional activity of the trapped gene. Poly(ADP-ribose) polymerase (PARP) inhibitors are the first DNA damage response targeted agents approved for cancer therapy. Y.I. Gene trapping has been used successfully to identify genes whose expression is induced upon in vitro differentiation of ES cells (16,43,44), growth-factor deprivation from a hematopoietic precursor cell line (13) and T-cell activation (14). Moreover, the RET system provides for rapid isolation of a cDNA fragment corresponding to an inducible gene by using 3′ RACE on chimeric NEO mRNA which is constitutively synthesized from an RNA polII promoter (Table 1). Here we report the construction and extensive examination of a versatile retrovirus vector, RET (removable exon trap). To examine more directly the effectiveness of our strategy for the enrichment of intragenic provirus integration events, we infected NIH 3T3 cells with the RET retrovirus and determined the nucleotide sequences of the 3′ untranslated regions (UTR) of various NEO mRNAs obtained from independently selected G418-resistant NIH 3T3 clones. Another important point is the fact that there is no significant difference in the proportion of G418-resistant colonies for the type III and IV viruses. We also thank Jeff Ecsedy, Jennifer Michaelson, Boris Reizis and Robert Weiss for their critical reading of the manuscript and Yaoqi Wang for expert FACS technique. Sugar modulation of anaerobic-response networks in maize root tips. Also, a high (>90%) success rate for the 3′ RACE procedure suggests that the majority of the NEO mRNAs derived from G418-resistant cells have poly A (or poly A-like) structures at their 3′ ends. In a similar experiment, the immunoglobulin κ-chain gene was found to be trapped by the RET vector in a GFP-negative mouse embryonic stem (ES) cell clone (data not shown), further supporting the notion that any gene can be trapped by the RET vector regardless of its expression pattern in the target cells. The type III and IV constructs possess, immediately downstream of the NEO cassette, a 1.2 kb SspI-EcoRV fragment of pKT3NP4 including the 3′ half of intron 8 and exon 9 (the last exon) of the mouse hprt gene. Insertional mutagenesis, in contrast, uses some form of a genetic tag to create mutations and subsequently recover the mutated genes. Insertion of a minimal disruption unit (the bcl-2 gene splice acceptor and the growth hormone gene poly A signal) into the intron appears to have completely abrogated the splicing and/ or transcription because only IL-4, and not AP, is detected in the supernatant of the transfected cells (Table 2).
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