Construction Of MWAP-hSA And MWAP-hFVII Hybrid Gene Locus And Evaluation For Their Expression Ability

The mammary gland bioreactor was an efficient expression system, in whichexogenous gene integrated into the host genome can be specifically expressed in themilk of transgenic animals.The expression vector was a critical factor for the mammarygland bioreactor. While there was not a versatility and efficient vector for the mammarygland bioreactor and it hindered development and application of this technology.To solve the above problem, a hybrid gene locus strategy was presented by us, inwhich the genomic coding sequence in the milk protein gene locus is replaced with thatof a foreign protein exactly from the start code to the end code. In other words, theforeign genomic sequence was flanked by the full5＇and3＇flanking region of the milkprotein gene locus in the hybrid gene locus,. We selected the mouse whey acidic protein(mWAP) gene locus and human lactoferrin (hLTF) genomic coding sequence to test thevalidity of our hypothesis. The results showed that rhLTF was highly expressed in themilk. The maximum expression level obtained was29.8g/L, three times higher ascompared with the best results reported by other group. In addition, the health oftransgenic mice was not affected in the condition of such high level expression ofrhLTF.The similar conduction of mWAP and human tissue plasminogen activator(htPA)gene locus emerged in the htPA transgenic mice. The highest expression level obtainedwas3.3g/L in the milk. It can be concluded that our hybrid gene locus combining thefull flanking regulatory element of milk protein gene locus and the target genomic DNAsequence can obviously ensure the extremely high level expression of exogenousprotein in the milk of transgenic animals.As a lactoprotein, the regulatory elements containing in the introns of hLTF genemay cooperate efficiently with the flanking elements of mWAP gene locus, whichensures the efficient transcription of hLTF gene. In addition, hLTF itself maybe isnaturally adaptive to be expressed and secreted in the milk. Besides, hLTF is a kind ofabundance protein highly expressed in the human milk. Considering thesecharacteristics of hLTF mentioned above, it may be not so remarkable for theachievement of hLTF expression employing our hybrid gene locus strategy. Otherexogenous proteins, such as those not naturally expressed in the mammary glandepithelial cells or which endogenous expression level is quite low should be chosen asmodels to test the broad application of our strategy. To this aim, the non-lactic butendogenous abundant protein, human serum albumin (hSA), and the endogenous low- level expression protein, human coagulation factor VII (FVII) were selected as targetproteins, corresponding mWAP-hSA and mWAP-hFVII hybrid gene locus wereconstructed separately and their expression ability were tested with transgenic mice asmodels. In addition to demonstrate the high performance and versatility of our hybridgene locus strategy, the transgenic mice expressing both of proteins represent anefficient system for the commercial production of rhSA and rhFVII.1. The construction of mWAP–hSA and mWAP-hFVII hybrid gene locusFor the construction of hybrid gene locus, at first, a gap-repair vector should beconstructed. A series of primers were design based on the guiding principle of no basepairs introduced or deleted. Then six400-to500-bp homologous arms (T1–T6) wereamplified by PCR with mWAP, hSA or hFVII BAC as the template, and these wereseamlessly ligated together in order and cloned into the modified pBR322vector(containing gene of promoter EM7and kanamycin). In the first step of the gap-repair,homologous arms T5and T6were dissociated by restriction enzyme digestion, and thelinearized vector was transformated into competent cells containing the mWAP BACclone and pKD46plasmid by electroporation. The pKD46plasmid expressed the Redsystem possessing gap repair. The8-kb3＇flanking region of mWAP was successfullygap repaired in this step. The second step gap-repair step started with the product of thefirst step: the arms T3and T4were dissociated, and transformated into bacteriacontaining the hSA or hFVII BAC clone and pKD46plasmid by electroporation, thenthe16-kb hSA or13-kb hFVII genomic sequence from ATG to TAA was gap-repaired.The third step of the gap-repair process started from the product of the second step: thearms T1and T2were dissociated and transformated into competent cells containing themWAP BAC clone and pKD46plasmid once again, and the13-kb5＇flanking region ofmWAP was gap repaired. Thus, the37-kb mWAP–hSA and34-kb mWAP–hFVII hybridgene locus were successfully constructed respectively, and the mWAP genomic codingsequence in the mWAP gene locus was exactly replaced by the hSA or hFVII genomiccoding sequence from the start code to the end code. PCR identification, Enzymedigestion and sequence analysis proved the mWAP–hSA and mWAP–hFVII hybrid genelocus to be the correct one.2. Expression and identification of rhSA and rhFVII in mouse mammary glandbioreactorThe hybrid gene locus fragment was released by Pvu I digestion and then was microinjected into fertilized C57BL/6J mouse eggs and the eggs transplanted intopseudopregnant females to generate corresponding transgenic mice. Total genomicDNA was prepared from a short segment of mouse tail to check for integration of theinjected DNA by PCR and Southern Blot. Totally seven transgenic mice weregenerated harboring the mWAP-hSA hybrid gene locus with33.3%positive ratio, andfifteen transgenic mice were generated harboring the mWAP-hFVII hybrid gene locuswith38.6%positive ratio.The expression of rhSA and rhFVII were identified by SDS-PAGE and WesternBlot analysis of milk samples. The results showed that five transgenic founders couldproduce rhSA or rhFVII respectively. The expression level of target proteins in milkwere determined by ELISA, and the highest expression level were11.9g/L for rhSAand49mg/L for rhFVII respectively. Further, transgenic copy number was determinedby real time PCR, and it was suggested that expression level of target proteins were notcompletely dependent on transgenic copy number. The expression level of rhFVII wasquite lower as compared with that of rhSA, we speculated that it's because of theinhibitory elements located in the first two introns of hFVII gene, in which high-copynumbers of repeated sequences are found, these elements may impede the efficienttranscription of rhFVII gene, and resulted in the much lower expression level ofrhFVII.Finally, tissue specificity of the transgenic transcription was determined byRT-PCR analysis in eight different tissues (mammary gland, heart, liver, spleen, lung,kidney, muscle and brain). The results showed that transcription of the mWAP-hSAhybrid gene locus was exclusively restricted in the lactating mammary gland in a waythat resembles the tissue specificity of the milk protein mWAP. The transcription of themWAP-hFVII hybrid gene locus was mainly found in mammary gland, but a muchweaker band also was illustrated in the brain tissue, it's still not so surprising because asreported, the mWAP gene is also marginally expressed in the brain tissue. Thebiological activity of mammary gland-synthesized rhFVII was determined usingAssaySense Human Factor VII (FVII) Chromogenic Activity Assay Kit and the activityof our rhFVII exceeded10.24IU/ml. This result demonstrated that our rhFVII possessthe natural biological activity of coagulation, which indicated that the rhFVII expressedin the mammary gland can be correctly modified after translation, especially can be carboxylated in multiple sites, a modification manner that is essential for the biologicalactivity of hFVII.In conclusion, the results indicated that the rhSA and rhFVII could be efficientlyproduced with our hybrid gene locus strategy. Together with our previous research onthe expression of rhLTF and rhtPA, we can draw the conclusion that the strategy isquite effective and versatility for the expression of exogenous proteins. Our researchprovide a new method for the construction of optimized expression vector for mammarygland.