Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene ...transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA. These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the α subunit of the human IL-3 receptor (hIL-3Rα) by factor-dependent growth. CD2 (frequency, 1 in 104) and hIL-3Rα (frequency, 1 in 1.5 x 105) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.
We have recently established a novel expression cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, ...pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.
DNA demethylation plays a critical role in transcriptional regulation in differentiated somatic cells. However, there is no experimental evidence that CpG methylation in a small region of a genome ...restricts gene expression. Here, we show that the anti‐CD3ε/CD28 antibody stimulation of human CD4+ T cells induces IL2 expression following epigenetic changes, including active demethylation of a specific CpG site, recruitment of Oct‐1, and changes in histone modifications. When the stimulatory signal is withdrawn, Oct‐1 remains on the enhancer region as a stable marker of the stimulation, causing the second induction to be faster and stronger. Our observations indicate that Oct‐1‐binding followed by CpG demethylation are key events in the epigenetic regulation of IL2 expression and may act as a memory of the regulatory event.
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