Split marker transformation increases homologous integration frequency in Cryptococcus neoformans
Split marker transformation increases homologous integration frequency in Cryptococcus neoformans
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DOI：
10.1016/j.fgb.2005.09.007
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发表时间：
2006-03-01
影响因子：
3
通讯作者：
Wickes, BL
中科院分区：
文献类型：
--
作者：
Fu, J;Hettler, E;Wickes, BL
由于异位整合和端粒化的高频率，新生隐球菌中的基因破坏可能是有问题的。为了提高同源整合的频率，采用称为分裂标记的转化策略，其利用由选择标记的重叠截短组成的DNA混合物。使用各种构建体比较五种基因的同源整合频率。当使用分裂标记方法时，同源整合率最高，取决于靶基因，同源整合率高达60%。导致同源整合频率增加的第二个因素是菌株背景，当使用双营养缺陷型作为宿主时，菌株背景最高。分裂标记策略与ura-blaster构建体相结合，该构建体已在其他真菌中用于回收ura 5或ura 3突变。当hisG-URA 5-hisG盒成功整合到靶基因座时，URA 5基因可以通过平板接种到5-FOA琼脂上而容易地被逐出。然后，该盒成功地用于第二个转化-驱逐循环。分裂标记破坏策略的有效性表明，对传统分子技术的持续研究和改进可以提高新型隐球菌分子操作的效率。(c)2006爱思唯尔公司All rights reserved.
Gene disruption in Cryptococcus neoformans can be problematic due to high frequencies of ectopic integration and telomerization. To improve the frequency of homologous integration, a transformation strategy was employed called split marker, which utilizes a mixture of DNAs comprised of overlapping truncations of the selectable marker. Five genes were compared for homologous integration frequencies using various constructs. Homologous integration was highest when the split marker approach was used, with rates as high as 60% depending on target gene. A second factor that contributed to an increased homologous integration frequency was strain background, which was highest when a double auxotroph was used as a host. The split marker strategy was combined with an ura-blaster construct, which has been used in other fungi to recycle ura5 or ura3 mutations. When a hisG-URA5-hisG cassette was successfully integrated at the target locus, the URA5 gene could be easily evicted by plating onto 5-FOA agar. The cassette was then successfully used for a second cycle of transformation-eviction. The effectiveness of the split marker disruption strategy suggests that continued investigation and modification of traditional molecular techniques could increase the efficiency of C neoformans molecular manipulation. (c) 2006 Elsevier Inc. All rights reserved.