Biomedical subjects

Jun-Tae Lee

Publications and source records attributed to Jun-Tae Lee.

3 recordsLinked to original sources

PubMed2007

Control of splicing efficiency by the mouse histone H2a element in a murine leukemia virus-based retroviral vector.

While using various human complementary DNA (cDNA) sequences in the context of the murine leukemia virus (MLV)-based retroviral vector, it was found that a retroviral vector containing some human cDNA sequences produces unusually low viral titer. One of those sequences is that for the human IL-1 receptor antagonist protein (IL1RN). The RNA analysis showed that a cryptic splice acceptor sequence is present in the middle of its coding region, resulting in the deletion of the packaging signal sequence and the removal of some coding sequences that lead to low viral titer and a low level of the transgene product. We tested whether the mouse Hist2h2aa1 element (mH2aE), previously shown to suppress the splicing function, could inhibit the cryptic splicing in the context of MLV-based retroviral vectors. It was found that the mH2aE could efficiently suppress such unwanted splicing event, thus increasing the amount of unspliced transcript, which eventually led to the increase in the level of IL1RN expression and viral titer. The mH2aE could also be used to control unusually high splicing activity. Our data suggested that the mH2aE could be used for the fine-tuning of the splicing process, thus improving the level of gene expression and viral titer in the context of retroviral vectors.

Base Sequence↗

PubMed2003

Construction of a retroviral vector production system with the minimum possibility of a homologous recombination.

A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia virus (MLV)-based packaging constructs contained significantly long 5' and/or 3' untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed.

Base Sequence↗

PubMed2002

MicroRNA maturation: stepwise processing and subcellular localization.

MicroRNAs (miRNAs) constitute a novel, phylogenetically extensive family of small RNAs ( approximately 22 nucleotides) with potential roles in gene regulation. Apart from the finding that miRNAs are produced by Dicer from the precursors of approximately 70 nucleotides (pre-miRNAs), little is known about miRNA biogenesis. Some miRNA genes have been found in close conjunction, suggesting that they are expressed as single transcriptional units. Here, we present in vivo and in vitro evidence that these clustered miRNAs are expressed polycistronically and are processed through at least two sequential steps: (i) generation of the approximately 70 nucleotide pre-miRNAs from the longer transcripts (termed pri-miRNAs); and (ii) processing of pre-miRNAs into mature miRNAs. Subcellular localization studies showed that the first and second steps are compartmentalized into the nucleus and cytoplasm, respectively, and that the pre-miRNA serves as the substrate for nuclear export. Our study suggests that the regulation of miRNA expression may occur at multiple levels, including the two processing steps and the nuclear export step. These data will provide a framework for further studies on miRNA biogenesis.

Biological Transport↗