Functional Analysis Of Chicken Ovalbumin Promoter

The chicken oviduct bioreactor plays an irreplaceable role in the production of pharmaceutic proteins due to its high capacity of egg production,quick scaleup of transgenic flocks,and similar glycosylation pattens to humans.Up to now,a number of transgenic chicken lines that produce a recombinant protein in egg white have been established.For example,interferon,lysozyme,antibodies,erythropoietin,and epidermal growth factor have been expressed and deposited in the egg white of transgenic hens.However,the transcriptional activity of ovalbumin promoters(OVPs)with different lengths,the regulation of estrogen response element(ERE)and the chicken ovalbumin upstream promoter transcription factors(COUP-TF)are short of deepening research.In addition,the technique for isolation and culture of chicken primordial germ cells(PGCs),or efficient carriers of a transgene,are still to be improved.The purpose of this paper was to find the core region of chicken OVP,and screen for the OVP with highest transcriptional activity,to explore the regulatory effects of ERE and COUP-TFs,and to establish a suitable culture method for chicken PGCs,and through in vitro and in vivo validation tests,finally to determine an promising OVP to be used in the preparation of oviduct bioreactors.1.Analysis of transcriptional activity of chicken ovalbumin promoter by CRISPR-d Cas9-VP64 systemThe promoters cERE-cOVP-2785 and cOVP-2785 were respectively subcloned to p GL3-Basic vector,and luciferase expression plasmids p GL3-cERE-cOVP-2785 and p GL3-cOVP-2785 were constructed.Firstly,HEK293 T cells were co-transfected with the aforementioned two plasmids plus p GL3 cOVP-1548 and plasmid p RL-TK by Lipofectamine 2000,and detected the to analyzed the transcriptional activities of the promoters were evaluated by detecting their luciferase activities.Using the Optimized CRISPR Design online,11 sg RNA and 2 sg RNA oligos had been respectively designed according to the DNA sequence of in the first intron and upstream of transcription start site(TSS)in chicken ovalbumin(OVA)gene,and that of the chicken ERE.11 p GL3-U6-OVP-sg RNA and 2 p GL3-U6-ERE-sg RNA recombinant plasmids were constructed respectively.HEK 293 T cells were co-transfected by single or multiple sg RNA expression plasmids with the combination of p GL3-cERE-cOVP-2785,and pc DNA-d Cas9 or pc DNA-d Cas9-VP64 plasmid in order to evaluate the effects of the promoter and ERE on luciferase expression via checking their own luciferase activities.The results showed that:(1)cERE-cOVP-2785 takes the first place in transcriptional activity,cOVP-2785 does the second;(2)d Cas9-VP64 plays a stronger activation effect on luciferase expression than d Cas9 does alone;(3)a stronger transcription activation effect is induced by multiple sg RNAs than a single sg RNA alone.(4)Each sg RNA in intron1,upstream of TSS and ERE regions could activate luciferase expression by targeting its own binding sequences.(5)The addition of estrogen to the culture medium significantly increases the expression level of luciferase.2.The functional analysis of the chicken ovalbumin upstream promoter transcription factor I/II(COUP-TFI/II).The recombinant plasmids named pc DNA3.1-COUP-TF? and pc DNA3.1-COUP-TF?were constructed.HEK293 T cells were co-transfected with pc DNA3.1-COUP-TFI and pc DNA3.1-COUP-TFII with combination of p GL3-cERE-cOVP-2785/p GL3-cOVP-2785 and p RL-TK plasmids using Lipofectamine 2000,the regulation effects of COUP-TFI/II on luciferase expression were assessed by testing their own luciferase activities.The results illustrated that:(1)the alliance of COUP-TFI/II and cERE-OVP2785 plays a upregulation of luciferase expression;(2)the combination of COUP-TF I/II and OVP2785 results in a down-regulation of luciferase expression.(3)Two transcription factors COUP-TFI and COUP-TFII play a synergistic role in regulating luciferase expression,and the regulation effect of COUP-TFII was stronger than that of COUP-TFI.3.Isolation and culture of chicken primordial germ cells(PGCs)Ficoll density gradient centrifugation and enzyme digestion method were respectively used to isolate PGCs from chicken embryonicbloodat HH-14 stage and gonads at HH-28 stage.The isolated PGCs were plated on BRL,STO and CEF feeder layers,and the proliferation capacities of PGCs derived from embryonic blood and gonads were compared,and their biological characteristics were determined.In addition,the cryopreservative effects of DMSO and ethanediol were evaluated.The results indicated that:(1)the proliferation capacity of PGCs derived from gonad(g PGCs)is superior to that of the PGCs from blood(b PGCs).(2)Being plated on BRL feeder layers,and cultured in the medium supplemented with 40% BRL conditioned medium and cytokines including SCF(6ng/m L),b FGF(4ng/m L)and LIF(10TU/m L),PGCs were survivable for more than 100 days,indicating this is feasible culture method for chicken PGCs.(3)Chicken PGCs were positive to PAS and AKP staining as well as to SSEA-1 immunofluorescence staining.Moreover,the PGCs transformed to embryoid bodies when cultured in differentiation medium.(4)10% ethandiol was the suitable cryopreservative medium for chicken PGCs.4.Functional verification of chicken ovalbumin promoter in vitro and in vivo293FT cells were co-transfected with the lentiviral vector p Lenti6.4/cEREcOVP-2785/c SLP-EGFP and the packaging plasmids ps PAX2,p MD2.G and p RSV-Rev using X-treme GENE HP DNA Transfection Reagent.The concentration of lentivirus was conducted by ultracentrifugation,lentivirus titer was determined by La SRT method.chicken OECs and g PGCs as well as 293 T cells were infected with the lentivirus,and the in vitro functional verification of cERE-cOVP-2785 was done through detection of EGFP expression level.Meanwhile,the lentivirus was injected to the dorsal aorta of chicken embryos at HH-14 stage,incubated in surrogate eggshells until to hatch.Subsequently,the in vivo fuctional verification of cERE-cOVP-2785 was performed via EGFP detection in gonads of chicken embryos with a fluorescent protein flashlight and matched filters,and via an investigation of EGFP integration to gonad genome using PCR.The results showed that(1)EGFP was expressed in chicken OECs and gonadal PGCs,but not in 293 T cells,indicating that the promoter is of tissuespecific.(2)EGFP not only expressed in gonads of chicken embryos,and but also integrated to the host genome,indicating that cERE-cOVP-2785 could effectively drive the expression of foreign genes in the gonad,but the stability of transgene expression needs further verification.In conclusion,the first intron and-54?-807 bp upstream of TSS in chicken ovalbumin(OVA)gene belongs to the core parts of ovalbumin promoter,and cERE-cOVP-2785 is a suitable ovalbumin promoter due to high transcriptional activity and the similar effect of ERE to a enhancer.The alliance of COUP-TFI/II and cERE-OVP-2785 plays an up-regulation of luciferase expression.Being plated on BRL feeder layers,and cultured in the medium supplemented with 40% BRL conditioned medium and cytokines including SCF(6ng/m L),b FGF(4ng/m L)and LIF(10TU/m L),is a suitable culture method for chicken PGCs.The promoter cERE-cOVP-2785 could effectively drive the expression of foreign genes in vitro and in vivo,indicating it is a promising OVP to be used in the preparation of transgenic chickens with oviduct-specific expression.