Mammary Gland-specific Expression Of HLF CDNA Driven By Lactoprotein And Cytomegalovirus (CMV) Chimeric Promoters

The use of transgenic animals for mammary bioreactors is a rapidly developing and intensely interesting field due to the increased demand for high-quality pharmaceuticals and medically diagnostic preparations.The manufacture of recombinant human proteins is more profitable by mammary gland bioreactor than that by other biological systems.The mammary gland-specific expression of a transgene is a major objective for producing heterogenous proteins in the milk of transgenic animals.Bio-active foreign proteins have been expressed specifically in the mammary under the direction of particular milk regulatory sequences,such as:goatβ-casein,bovineαs1-casein,ovineβ-lactoglobulin (BLG) and rat whey acid protein(WAP).However,much of the evidences regarding the mammary as bioreactor indicated that only a small percentage of the mammary glands of transgenic animals expressed functional protein,and most of them were found at low levels in milk.Several major regulatory elements are required in order to obtain satisfactory transgene expression,such as essential gene insulators,chromatin openers,enhancers,matrix attached regions,and introns.One of the most important elements may be the factor controlling the transgene's transcription and translation.In recent years,although lactoprotein gene promoters were widely used,it should be noted that their expression levels in milk were relatively low and unstable in many instances.As an alternative to the widely used cytomegalovirus(Pcmv) enhancer for expressing transgene in eukaryon,there was no report for promoting transgenic expression in mammary gland,our aim was to build a series of promoters bearing at least one,or if possible,all of the following characteristics:promoters that would increase high levels of expressing transgene in milk,and expression specifically located in the mammary organs. We used the chimeric promoters/enhancers combining sequences of lactoprotein promoters from cow and goat,with the Pcmv or mono-milk-promoter without Pcmv or SV40ployA to construct vectors for expressing human lactoferrin(hLF) cDNA,their ability to drive high levels of specific recombinant human lactoferrin(rhLF) expression was determined in transgenic mice or goat mammary epithelial cells(GMECs) in vitro.Vectors of mono-promoters of lactoprotein elements were constructed as a control.Seven vectors (BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36) with different promoters/enhancers were constructed.Three vectors of BnF95,pBnCL14 and pBnLC2G were constructed by using milk regulatory elements of goat BLG and neomycin resistance marker gene(NEO^r) for transfecting of GMECs;pgCNCG/LF25,and pbCNCS/LF36 were constructed by using promoter enhance sequences of goat casein, bovineαs1-casein,chickenβ-globin insulators,Pcmv and SV40polyA for producing transgenic mice.pgCN/LF/g and pbCN/LF contained mono-promoters of casein as controls.Primary GMEC lines were derived from biopsy tissue from artificial lactating goats. The cells were cultured in DMEM/F₁₂ containing 10%fetal calf serum(FCS).GMECs could keep proliferate vigorously even 25 passages in vitro.The pure GMECs lines could be obtained within 2-3 passages by multi-alternation of digesting and adhering repeatedly. By electroporation,BnF95,pBnCL14 and pBnLC2G encoding neomycin resistance for selecting the positive cells were transfected into GMECs in vitro.After three-weeks-culture by G418(400ng·L^-1),the transfected cell colonies grew and proliferated in G418 medium. Single colonies were picked into another 24-well plate for continue culture,and 242 colonies were generated from 118 clonies of GMECs by neo-resistant culture.Three of cell clones from 242 colonies were respectively screened by PCR and each was confirmed to be transgenic colonies corresponded with NEO^r ones,the total 82,42 and 118 colonies were transfected transgenes of pBnCL14,BnF95 and pBnLC2G respectively.These colonies were induced with luteotropic hormone,and then the media were gleaned respectively at 48h or 72h.It was found that there were 30 colonies expressing recombinant human lactoferrin(rhLF) by ELISA at 72h.4 colonies of higher expression level of rhLF were about 50mg·L^-1 from transgene of pBnCL14.These GMECs of expressing rhLF would be used as donor cells for hircine somatic cell nuclear transfer(SCNT) to produce the transgenic goats.Four vectors(pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36) were used to generate transgenic mice.A total of 14 lines were selected out of 160 candidates.To verify transgenic integration,these PCR products were sequenced,and the sequences were analyzed using DNAStar software.Compared with correlative transgenes,the percentage of sequence identity was 100 except 99.75 for pgCNCG/LF25 founder line.Founder mice bearing the transgene were mated to C57 males or female and delivered 110 pups,of which 52 progeny were identified as being transgenic.Using ELISA detection,results showed that the recombinant human lactoferrin rhLF expression was significantly increased in the milk of transgenic mice with chimeric promoter transgenes.The expression level was 2-8.1g·L^-1 in the milk using chimeric promoter.In contrast,only 0～0.012g·L^-1 was found in the milk using mono-casein promoter.In two of the highly expressing lines,pbCNCS/LF36 and pgCNCG/LF25,there was approximately a 10,000-fold increase in target protein in the milk of transgenic mice (P＜0.001).In addition,we have also shown that the expression of rhLF was strictly limited to the mammary organs in both founders and F1 offspring,because no rhLF was detected in the saliva or blood of transgenic mice expressing high levels rhLF in milk.This result suggested that the constructs with chimeric promoter/enhancer could increase the expression of heterogenous protein in the mammary gland of transgenic animals with high specificity.Recently,continuing discoveries of new and surprising mechanism of gene regulation suggest many and perhaps all genes are regulated at multiple steps including transcription, post-transcriptional processing,nuclear export and localization,stability,and translation of mature mRNA molecules.Chimeric promoter/enhancer contains both mammary gland specific elements and Pcmv for mammary expression.Bio-informatics analysis shows that Pcmv sequence contains 10 uORF(upstream open reading frame) and 4 CCAAT/enhancer sites.Lodhi et al(2003) considered that the uORF could mediate constitutive effects on translation of mRNA from AUG codon.CCAAT sequence is an enhancer-binding site having been described in being linked to CCAAT-binding protein,some research show the increasing of the binding would mediate expression of functional gene,which might underlie observation that some cis-acting elements within the 5'-UTR including chimeric promoters of lactoprotein and CMV can activate initiation of translation.From overall results,pgCNCG/LF25(composed of Pcmv chickenβ-globin and goatβ-casein promoter),pbCNCS/LF36(Pcmv,SV40polyA and bovineαs1-casein promoter),pBnCL14(Pcmv goatβ-BLG promoter AND SV40polyA) and pBnLC2G (Pcmv,2×chickenβ-globin and goatβ-BLG) were the most efficient under in vivo and in vitro conditions.The pBnCL14 was the most efficient in BnF95 in the expression of GMECs in three of pBnCL14,pBnCL14 and pBnLC2G,while the PgCNCG/LF25 was more efficient than the pbCNCS/LF36 in mammary gland of transgenic mice.It is worth mentioning that the binary promoter of Pcmv can improve mammary gland-specific expression,while ternary promoter of CMV,chickenβ-globin and lactoprotein may be more active than the binary one in mammary gland of transgenic mice.Conclusively,although many long regulatory sequence can be useful for driving expression of targeted transgene in milk,their low efficiencies and uncertain expression,in most transgenic individuals,caused researchers to concentrate much time and effort on constructing over-expressing vectors for mammary specific-gland expression.Compared to constructing large size regulatory elements,the chimerc promoters/enhancers for conveniently constructing mammary-specific expression vectors,were shown to ascribe a high level of stability and efficiency of expression.We describe for the first time the synergistic effect achieved by the use of binary or ternary promoters/enhancers for mammary specific expression of rhLF in transfected GMECs and the milk of transgenic mice.