Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation
Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation
复制标题
使用改造用于植物转化的 YAC 将大 DNA 片段基因枪转移至烟草细胞
作者：
J. Mullen;G. Adam;A. Blowers;E. Earle
To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.
DOI：
10.1073/pnas.87.13.5109
发表时间：
1990-07
影响因子：
11.1
作者：
V. Pachnis;L. Pevny;Rodney Rothstein;Frank Costantini
通讯作者：
V. Pachnis;L. Pevny;Rodney Rothstein;Frank Costantini