Chatterjee Pradeep K, Shakes Leighcraft A, Stennett Naima, Richardson Vanessa L, Malcolm Tennison L, Harewood Ken R
Department of Chemistry, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.
BMC Res Notes. 2010 Feb 19;3:38. doi: 10.1186/1756-0500-3-38.
Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.
A general procedure to replace the loxP site located at one end of genomic DNA inserts in BACs with lox66 is described. Truncating insert DNA from the loxP end with a Tn10 transposon carrying a lox66 site simultaneously substitutes the loxP with a lox66 sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of loxP with lox66 or lox71 was found to be as efficient as another loxP site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a lox66 transposon occurred at no more than 20% the efficiency observed with a loxP transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical lox-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.
The loxP/lox66 replacement procedure should allow targeting BACs to a pre-positioned lox71 site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.
与转基因整合位点相邻的染色质会影响基因表达，使得对密切相关的转基因（如那些通过增强子捕获改造的BAC缺失系列衍生的转基因）进行比较变得不可靠。将基因靶向染色体上的预定位点可能会缓解这一问题。
描述了一种用lox66替换BAC中基因组DNA插入片段一端的loxP位点的通用方法。用携带lox66位点的Tn10转座子从loxP端截断插入DNA，同时用lox66序列替换loxP。这种替换具有高严谨性，该方法应适用于公共领域的所有BAC。在含有loxP和lox66或lox71位点的小质粒的噬菌体P1转导过程中，发现loxP与lox66或lox71的Cre重组效率与另一个loxP位点相同。然而，使用lox66转座子对BAC中的插入DNA进行末端缺失的效率不超过使用loxP转座子观察到的效率的20%。P1生命周期不同阶段可用的Cre蛋白重组相同与不同lox位点能力的差异可能是造成这种差异的原因。讨论了解释这些发现的一种可能机制。
loxP/lox66替换程序应能将BAC靶向斑马鱼染色体中预先定位的lox71位点；在该系统中，同源重组介导的“敲入”技术不可用。
