Research On Construction Of BAC Library Of Guanzhong Milk Goat And Homologous Recombination With Red System

A new vector system-BAC(bacterial artificial chromosome) has been developed into an important method for the construction of complex genomic library. BAC library plays a pivotal role in genome studies, and modification of BAC also has an important role for analysis and studying gene function and expression in vitro and in vivo. The homologous recombination technology with red system amplifies the application of BAC library, and provides a new path of eliminating position effect of random intrgration in transgenic animals. The goal of the research is constructing of BAC library of Guanzhong milk goat genome DNA which has high milk efficiency, and substituding encoding sequence of native ht-PA to encoding sequence of β-casein with red system, promoting the expression of native ht-PA by β-casein controling sequence, overcoming the position effect of random integration in transgenic animals, to provide an efficient vector for preparation of mammary gland bioreactors which expresses native ht-PA. This dissertation is divided into two segments:1. The former segment separated large DNA in CHEF(contour-clamped homogeneous homogeneous electric field), and extracted DNA with electroelution, the received DNA fragments were ligated to pBeloBAC11 digested by the restriction enzyme of BamHI and dephosphorylated by CIAP phosphatase, transformed competent cell DH10B by electroporation of large DNA, acquired the part BAC library of Guanzhong milk goat, whose average inserted fragments ranged from 30 kb to 40 kb. We explored many factors which influenced high efficient transformation of large DNA, such as prepartion of BAC invector and high-molecular-weight DNA, partial digestion, ligation, electroporation, ,and provided a basis of construction of the whole library. 2. The latter segment construced two native ht-PA vectors with carrying zeocin resistance gene, native ht-PABZeo and native ht-PAZeo, one contained BGHpA, another not. Zeocin resistance gene has eukaryote and prokaryote promoters, so it is used to screen by resistance in both mammalian cell lines and bacteria. PCR fragments of above two vectors were electro-transferred into competent cell DH10B, promoted homologous recombination with the role of red recombinant system, the encoding sequence of β-casein was in vivo replaced with native ht-PA encoding sequence. BAC clones were identified by PCR and sequence analysis, and proved that native ht-PA was integrated into β-casein location as expected.