Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination.
Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination.
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DOI：
10.1093/nar/28.18.e84
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发表时间：
2000-09-15
影响因子：
14.9
通讯作者：
Ioannou, P A
中科院分区：
文献类型：
--
作者：
Orford, M;Nefedov, M;Ioannou, P A
GET重组是一种简单的大肠杆菌诱导同源重组系统，用于在200 kb BAC克隆的β -珠蛋白基因的起始和终止密码子之间插入EGFP盒。β -珠蛋白和δ珠蛋白基因启动子区域之间的高度同源性也允许同时产生δ珠蛋白报告基因构建，并删除8.8 kb的中间序列。两种构建体在短暂转染MEL细胞后均表达EGFP。同样，靶向γ -珠蛋白基因启动子区域和β -珠蛋白基因终止密码子之间的EGFP盒，可以生成(A) γ -珠蛋白和(G) γ -珠蛋白基因的报告基因构建体，分别涉及24和29 kb基因组序列的特异性缺失。最后将EGFP盒插入到epsilon-和-球蛋白基因之间，同时删除44 kb的中间序列。这些修饰结构的生成效率很高，说明了GET重组在功能研究中产生BACs中特定序列的大量缺失的有效性。建立具有这些珠蛋白结构的稳定的红细胞细胞系将有助于寻找以生理相关方式改变单个珠蛋白基因表达的治疗剂。
GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.