Studies On Expression Of Human-lactoferrin CDNA Under The Multiple Promoter/enhancer Of Bovine αs1 Casein/CMV

Many scientists devoted to study mammary gland bioreactor of human lactoferrin by transgenic animals, which provides the possibility to produce rhLF for medical application characterized by high quantity and low cost.However, the expression level of hLF is low in animal mammary gland. To obtain high and stable expression of hLF in transgenic animals, various promoters used to construct expression vectors in this research. Two vectors were constructed to express hLF cDNA in transgenic animals. CMV enhancer is the different point between the two vectors. Evaluating the efficiency of CMV enhancer in transgenic expression, and screening the homozygous transgenic mice by hybridization, which laid the foundation for producing transgenic goat mammary gland bioreactors.We used the bovineαs1-casein regulatory sequence, it conclude 5'flank regulatory region(2264bp length,cover the forward region of second exon, first intron, first exon,the upper 818bp sequence of 5'flank), 3'flank regulatory region(1440bp length, cover the last exon,the last intron, 3'terminal non-translation region), and the upstream 4kb(form -2264 to -6264). We use hLF cDNA and enhancer DNA fragment. Enhancer sequence was obtained by commercial construction. Then, we acquire two constructions, AF and AP,which is contain CMV enhancer sequence from the former.Transgenic mice were generated by pronuclear DNA microinjection using AF and AP of the two constructs. The number of mice we obtained is 56 and 51 seperately. 851 microinjection embryos were transferred into 20 recipients. 56 AF offsprings were obtained, the rate of survive embryos is 57.6%.Then, 630 microinjection embryos were transferred into 37 recipients. 51 AP offsprings were obtained, the rate of survive embryos is 42.3%.A total of two female AP transgenic mice(42#, 46#) were identified by PCR and sequencing. The efficiency of transgene integration was 3.92%. All transgenic-positive mice grow and reproduce offsprings as normal as wild mice.We obtained 5 F1-offspring and 7 F2-offspring transgenic mice,then we generated transgenic mouse lines.The result of ELISA assay and Western blot showed that the milk of the G0 transgenic mice had expressed hLF and the level reached 3⁶ g·L^-1, and its offsprings also had expressed hLF.Otherwise, the serum and saliva of transgenic mice was checked.Compared with normal mice, level of hLF in blood of 42#, 46# mice had no increase during lactation, suggesting the mammary gland-specific expression of this construct.For 56 AF mice, we checked their latex and detect them in atomy medical institute of Jiangsu Prov, which had been harboured in our laboratory.Through hybridization,we obtained 2 homozygous(1♀1♂). Through ELISA assay, hLF protein was measured.The level of hLF expression in transgenic mice of AP vector was higher than AF vector. The result confirmed our presumption, The cDNA, CMV and the regulatory sequence of bovineαs1 casein were assembled into mammary gland specific construct, The modified hLF version was highly expressed in mammary gland of resulting transgenic mice. The expressive specificity,stability,profile and bioactivity of this hLF were analyzed. The results of this research paved the way to produce mammary gland bioreactors of transgenic goat.