[Establishment of BAC mediated transgenic mice containing 97 kb beta-globin gene cluster].
OBJECTIVE
To delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster.


METHODS
Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice.


RESULTS
By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines.


CONCLUSIONS
Human beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.
Shen
Wei
W
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS, PUMC, Beijing 100005, China.
Huang
Yue
Y
Tang
Yi
Y
Liu
De-pei
DP
Liu
Guang
G
Wu
Min
M
Liang
Chih-chuan
CC
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004247:DNA; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D017370:Interleukin-11 / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D005810:Multigene Family / Q000235:genetics*; D018123:Receptors, Interleukin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
D017370:Interleukin-11; D018123:Receptors, Interleukin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005914:Globins; D004247:DNA