title: Variable expression of human lactoferrin gene in mice milk driven by its 90 KB upstream flanking sequences.
abstract: One major drawback in research of animal mammary gland bioreactors is the low production rate of high-expressing transgenic animals due to position effects. To obtain high and stable expression of foreign gene, yeast and bacterial artificial chromosome have been used as transgene vector in recent research. Human lactoferrin is a bioactive, versatile protein, and has large potential in nutritional and therapeutic applications. Therefore, production of recombinant lactoferrin using animal bioreactors was studied widely to satisfy its large requirement. We reported here a transgenic mice model with high-level expression of recombinant human lactoferrin in mammary gland. Transgene construct used here was a human bacterial artificial chromosome containing intact lactoferrin-encoding transcript unit, approximately 90 kb 5'-flanking sequences and 27.2 kb 3'-flanking sequences. We obtained totally 10 transgenic mice whereas two of them lacked of part of upstream sequences of the gene. Milk of eight transgenic mice line was detected by Western blot and radioimmunoassay and seven lines expressed recombinant human lactoferrin at high but variable level (0.29, 0.53, 0.90, 1.23, 2.76, 3.58, and 8.02 mg/mL, respectively). The variability of expression indicates that even the 90 kb 5' flanking sequence of the transgene can't overcome position effects completely. Moreover, we also determined sequences of 9.3 kb regulatory region and 10.6 kb encoding region of the gene and thus supplemented all unknown sequences. Our results suggested that transgene vector used here has potential to be used in large farm animals for production of recombinant human lactoferrin in industrial scale.
authors.affiliation: State Key Laboratories for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, PR China.
authors.forename: Zhaoliang
authors.initials: Z
authors.lastname: Liu
authors.forename: Chunjiang
authors.initials: C
authors.lastname: Zhao
authors.forename: Baoliang
authors.initials: B
authors.lastname: Fan
authors.forename: Yunping
authors.initials: Y
authors.lastname: Dai
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Zhao
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Zheng
authors.forename: Jidong
authors.initials: J
authors.lastname: Feng
authors.forename: Yizhen
authors.initials: Y
authors.lastname: Chen
authors.forename: Yingli
authors.initials: Y
authors.lastname: Duan
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007774:Lactation / Q000235:genetics* / Q000378:metabolism; D007781:Lactoferrin / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D008321:Mammary Glands, Animal / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D007781:Lactoferrin