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Our major research interests are intracellular transportation and recombineering. Within the cell, a variety of components are moved to specific sites at specific times. The intracellular transportation processes are essential not only for housekeeping purposes but also for specialized cellular functions, such as the transport of synaptic vesicles. We use transgenic and knockout mice to study the functions of kinesin-1 (a key transporter molecule) in chondrocytes, neurons, and other cell types for its roles in development, cellular function, and genetics. We're also interested in identifying cargo proteins carried by kinesin-1 in different cells. Recombinogenic engineering methodology, also known as recombineering, uses homologous recombination to create targeted changes in cellular DNA with great efficiency, specificity, and flexibility. We have investigated the mechanisms underlying recombineering and improved its efficiency. The laboratory is currently applying recombineering to the systematic studies of small noncoding RNAs, proteome localization, and biotech development.
www.hku.hk/biochem/biochem/pi/pi_jdhuang.htm
Our laboratory frequently needs to construct various plasmids and BAC vectors. In our paper, we focus our study on single-stranded oligonucleotide-mediated deletion on plasmid DNA. We found that a single-stranded oligonucleotide as short as 63 nucleotides was sufficient to mediate deletion of as long as 2 kb with efficiency higher than 1%. We provided an optimized protocol for single-stranded oligonucleotide-mediated deletion modification of plasmid DNA. This protocol is so efficient that plasmids can be manipulated without selectable markers. The procedure is very simple. To delete a sequence in a plasmid, first, an oligonucleotide is designed with its 5′ and 3′ halves homologous to the flanking regions of sequence to be deleted, respectively. Second, the oligonucleotide is transformed into recombinogenic competent cells harboring the target plasmid. Third, several bacterial colonies are picked up to perform colony PCR with PCR primers flanking the region to be deleted. We can usually obtain 5 modified clones by screening 100 colonies.
See “Highly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides” on page 217.
