George Searfoss

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml−1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml−1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.

hUBC9 associates with MEKK1 and type I TNF-α receptor and stimulates NFκB activity

hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two‐hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF‐α receptor, respectively. Because both of these proteins regulate NFκB activity, the role of hUBC9 in modulating NFκB activity was investigated. Overexpression of hUBC9 in HeLa cells stimulated the activity of NFκB as determined by NFκB reporter and IL‐6 secretion assays. hUBC9 also synergized with MEKK1 to activate NFκB reporter activity. Thus, hUBC9 modulates NFκB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF‐α receptor.