TransgenicChickenswithoviductspecificvector.doc

Transgenic Chicken with Oviduct Specific Vector AbstractThe present study was attempted to replace ovalbumin gene with tissue plasminogene activator marked with green fluorescence protein. The pl-EGFP, pl-CMV-tPAEGFP and pl-2.8OV-tPAEGFP vectors were constructed. 293FT cells were transfected with pl-EGFP vector and chicken embryo fibroblasts (CEF), chicken primary germ (cPGC) cells with pl-CMV-tPAEGFP vector. The green fluorescence protein was expressed among all trasnfected cells. The pl-2.8OV-t-PAGFP and control (pEGP-N1) vector was transfected with Hela, C127 and oviduct epithelial cells. The pl-2.8OV-t-PAGFP vector was only expressed in oviduct epithelial cells but not in hela and C127 while control vector was expressed in all hela, C127 and oviduct epithelial cells. The result showed that pl-EGFP, pl-CMV-tPAEGFP vectors could infect different cells but pl-2.8OV-t-PAGFP vector was only expressed in Oviduct epithelial cells due to oviduct specific expression system. The lentivirus with (pl-2.8OV-tPAEGFP) was injected in 50 fertilized eggs, 11 (22%) chicken hatched with 4 (36%) positive containing integration of exogenous genes analyzed by DNA dot blotting and PCR. Our finding suggested that transgenic chicken can be produced using oviduct specific vector containing pl-2.8OV-tPAEGFP.Key Word: Lentiviral vector; ovalbumin gene; tissue plasminogene activator; GFP; laying hensIntroductionPresently one of the most advanced research of is genetically modified technology, it has been involved in production of transgenic animals. The rst transgenic animals were generated by infecting mouse blastocyst cells with a Moloney leukemia virus-based retroviral vector (Jaeisch 1988, 1976). Lentiviral vector has been used successfully to create transgenic mice with a very high rate of incorporation of the foreign gene (Lois et al., 2002). Lentiviral vectors allow for high levels of transgene expression in quails and chickens (Benjamine and Carlois 2006) Tissue-specic expression in transgenetic birds (PNA Sin press). Tissue plasminogen activator(tPA), a major protein involved in the breakdown of bloodclots, to treat heart attacks, strokes, clots in the lungs and is also being studied in the treatment of cancer. As anenzyme, it alsocatalyzesthe conversion ofplasminogentoplasmin, the major enzyme responsible for clot breakdown. tPA has also been used to dissolve thrombi associated with ischemic strokes and brain injury, people withfrostbitewho were treated with tPA had feweramputationsthan those who were not (Bruen et al 2007, Tsurupa and Medved (2001) , Ichinose et al (1986) Keeping in views the importance of tissue plasminogene activator , we have used recombinant human tissue plasminogene to replace ovalbumin gene in magnum of oviduct of laying hens. Ovalbumin is the major protein of the magnum of the hens oviduct and is not normally found in other tissues (Kohler et al 1968）. The chicken ovalbumin gene encodes more than half of egg white protein， with approximately 2.2 g per egg (Burley and Vadehra, 1989). The oviduct of laying hen has approximately 105 copies of the ovalbumin mRNA per cell (Palmiter, 1975). Ovalbumin constitutes 54% of the protein in the egg white and its steroid-responsive tissue-specific regulation is well documented (Dougherty and Sanders, 2005). We constructed pl-2.8OV-T_PAGFP vectors by cloning oviduct specific promoter from ovalbumin gene of laying hens and human tissue plasmingene activator.Materials and Method增強型綠色熒光蛋白基因（ EGFP ）來自 pEGFP 質(zhì)粒 (GeneBank Accession No: U55762 本室保存 ) ，由 pEGFP 質(zhì)粒經(jīng) Nde 、 Xba （ NEB 公司）雙酶切后經(jīng)瓊脂糖凝膠電泳分離純化后獲取pEGFP plasmid (already saved in our laboratory and the pL-lacZ plasmid (Invitrogen) containing (lentiviral vector sequence) were digested by Nde 1, Xba 1 enzymes and the new plasmid named pL-EGFP . The newly constructed plasmid was載體去磷酸化及快速連接方法參見相關(guān)產(chǎn)品附帶說明。 purified by相同的酶酶切 pL-lacZ 質(zhì)粒（ Invitrogen ）獲取含慢病毒轉(zhuǎn)運載體序列的 DNA 片段，以熱酶磷酸酶進行載體去磷酸化（ NEB ），瓊脂糖凝膠電泳分離純化后以快速連接試劑盒（ LigaFastTM Rapid DNA Ligation System, Promega ）與 EGFP 連結(jié)，最后轉(zhuǎn)染 JM109 感受態(tài)細菌擴增質(zhì)粒，新質(zhì)粒命名為 pL-EGFP （圖 1-1 ）。 Quick Connect Kit (Liga Fast TM Rapid DNA Ligation System, Promega).Fig 1-1 The construction of pL-EGFP plasmid The tissue plasminogen activator gene with pGEM-tPA plasmid was received from Professor Tao Xi, China Pharmaceutical University. For the insertion of tissue plasminogene activator into the pL-EGFP vector, t-PA primers were designed with sense primer 5GCGGGAATTCTCGAGATGGATGCAA3 and Antisense Primer 5GATTATCACGGATCGATGTTGTCAC3 with Xho1 and BamH1 restriction site. The 20L PCR reaction system to include: 50ng genomic DNA, 2l PCR buffer, 0.2 mM dNTPs, 10M primer, 0.5 units of Taq DNA polymerase (Promega).PCR reaction conditions : 94 denaturation 2 min; 94 30s, 58 30 s and 72 2 min total of 32 cycles, final extension 72 for 7 min. The pL-EGFP- plasmid was double digested with Xho1 and BamH1 and PCR products was sub cloned in it and the resultant vector was named pL-tPAGFP plasmid. The 500 ul of blood was collected from Isa Brown breeds by wing vein and its DNA was extracted using the trizol protcol (invitrogen Kit with cat. No 15596-026. Primers sense P1：5CATCTTGTCATATGTCCTCAGACTTGGC3 and antisense P2：5GAGCCTCGAGTGAACTCTGAGTTGTCTAG were designed with restriction enzyme sites Nde1 and Xho1 respectively, for amplification of 2.8kb fragment of chicken ovalbumin gene. The 50Ul of PCR reaction contained 30uM each primer, 500ng of chicken genomic DNA, 200uM of dNTP and 2.5U of Pfu DNA polymerase (promega, Madison, W1). After denaturation at 94 for 4 minutes, 30- cycle PCR was performed using the following program: denaturation at 94 for 30 second, annealing at 60 for 30 second and elongation at 72 for 3 minute (7 minutes for final cycle). PCR product with 2.8 kb sequences was inserted into pL-tPAGFP vector using Nde1 and Xho1 enzymes. The insertion of ovipromoter resulted in the deletion of CMV promoter and newly resultant vector was called Pl-2.8OV-tPA GFP. Preparation of Plasmid DNASingle colonies of E. coli transformants of the each plasmid vector were grown overnight in 500 ml of Luria Bertani (LB) broth containing 100ug of ampicillin /ml. The plasmid DNA was prepared using standard alkaline lysis method, Purified by the PEG (BBI, Toronto, Canad), precipitation (1) Culture of cells293FT, Hela, 293FT, C127, chicken embryo fibroblasts (CEF), chicken primary germ cells (cPGC) were cultured accordingly our laboratory method. The finally minced oviduct tissues were several times washed in phosphate buffered saline (PBS and finally suspended in DMEM containing 10mmol/L HEPES as well as collagenase at a concentration of 0.5mg/ml. The enzymatic disintegration of the oviduct was performed in a 100ml glass bottle in a total volume of 20ml by incubating the vessel for 1 h in a shaking bed at 37 C. The tissue residue was removed by filtering the dissociation mixture with layers of gauze. Epithelial cells were collected by centrifugation at 600 rpm for 2 min. The cells were washed two times with DMEM containing 10% fetal bovine serum, 50g/ml penicillin, 50g/ml streptomycin then the primary oviduct cells were transfected immediately and incubated at 37 C or 41 C, 5% CO2 in DMEM supplemented with 10mmol/L HEPES at pH 7.4, and antiobiotics, 8% chicken serum, 2% fetal calf serum, 710mol/L 17-estradiol (Sigma,( ) USA), 6-10mol/L corticosterone (Sigma, USA), and 50g/L insulin (Sigma, USA).In vitro DNA transfection393FT cells were transfected with Pl-EGFP vector; 293FT, C127, chicken embryo fibroblasts (CEF), chicken primary germ cells (cPGC) cells with pl-t_PAGFP vector and primary oviduct cells were transiently transfected by polyethyleneimine procedure. Four microlitre 10 mmol/L polyethyleneimine was added to two micrograms of each vector respectively and mixed, and then cultured for 15 min at room temperature. Then each mixture was pipetted into 106 cells plated on a 35 mm plastic culture dish in 1 ml of basal medium DMEM supplemented with 1% fetal calf serum. After the cells were cultured at 37 C to 41 C in 5% CO2 for about 4 h, the medium was changed with DMEM supplemented with 10 mmol/L HEPES at pH 7.4, 8% chicken serum, 2% fetal calf serum, and antibiotics 7-10 mol/L 17-estradiol, 610 mol/L corticosterone 50 g/L insulin. The cells were examined at 48 h after transfection. Green fluorescent protein was checked for confirmation of vector. Through fluorescence microscope the green fluorescence protein was checked among the cells. Lentivirus production To prepare lentiviral stock 293FT cells were plated on 144-mm dishes with 3.5ug of vector plasmid, 5ug of gag/pol plasmid, 2.5ug of rev plasmid and 3ug VSV-G plasmid by lipofectamine TM 2000. Forty eight hour after transfection, tissue culture medium was harvested and filtered (0.22um), then centrifuged at 6,000 ug for 20 h at 4C, followed by ultracentrifugation at 50,500ug for 120 min at 4C.The pellet was resuspended in formulation buffer (20mMTris/100mM NaCl/10mg/ml sucrose/10mg/ml mannitol,pH7.4) . A liquots of vector were stored at 80C.Egg injection50 fertilized eggs were positioned with their sides facing upwards for 8 h at room temperature in order to fix the blastoderm position. After swabbing the shell with 70% alcohol, 4.5 x 4.5mm2 sized windows was made in the equatorial plane of the egg shell using a fine drill, which was followed by removal of the small shell membrane inside the window with fine forceps and a surgical blade . Five micro liters of DMEM containing concentrated virus with pl-2.8OV-t-PAGFP was injected into the central part of the sub germinal cavity using a microinjection pipette. To increase infectivity, polybrene (10ug/ml) was added to the virus medium. The injection pipette was drawn from a Pyrex glass tube with inner diameter at the tip of 80 um. After injection, the window was sealed with egg shell and paralm.Egg incubation After microinjection, the sealed eggs were incubated at 37.5 C and 60 % relative humidity with a rocking motion every 2 h through a 90 angle for 18 days, following which they were further incubated at 37 C and 70 % relative humidity without rocking until hatching .The age of an egg is based on days post-incubation (e.g., the day of microinjection is referred to as a day0).Genomic analysisGenomic DNA was extracted from chicken using genomic DNA purication kit (Promega). 20L PCR reaction system to include: 50ng genomic DNA, 2l PCR buffer, 0.2 mM dNTPs, 10M primer, 0.5 units of Taq DNA polymerase (Promega). PCR reaction conditions : 94 C denaturation 2 min; 94 C 30 second ; 58 C 30 second and 72 C 2 min total of 32 cycles , final extension 72 C for 7 . In order to achieve rapid results the presence of transgene was screened using the DNA dot blot analysis. Briefly 20ug of DNA was cut with ultrasound and dropped on a nylon membrane. 1700bp probes tissue plasminogene activator probes for blot analysis were amplified with the primers 5GCGGGAATTCTCGAGATGGATGCAA3 and reverse 5GATTATCACGGATCGATGTTGTCAC3 purified by agarose gel electrophoresis and then synthesized using PCR DIG probes synthesis kit (Roche).ResultPl_Egfp vector The pl-Egfp constructed in this study is given below in figure 1. In figure 1, (A) is the agarose gel analysis of pL-GFP plasmid, Supernatant of rapidly SDS-alkaline lyses bacterial cells was directly loaded and only supper coiled band was detected. The 1 and 2 are the samples for plasmid size and SM, supper coiled marker (TaKaRa). The B and C are results for Double digestion of pL-GFP plasmid by restriction enzyme analysis. In figure B the 1 and 2 are the samples for digestion result of vector and M： marker (TOYOBO) While C is the diagram from the analysis of Gene Construction Kit 2.0. M： marker (TOYOBO) Fig 1, Construction map of vector, size and digestion resultsFor pl-eGFP plasmid confirmation, 293FT cells were transfected with pl-eGFP vector and EGFP was expressed in these cells. Fig 2 shows the expression of green fluorescence protein in 293FT cells. A is image for green fluorescence protein and B bright field image of A Fig 2, 293FT cells transfected with pL-EGFP plasmids by LipofectamineTM 2000, A green fluorescence image and B bright field image of A.pl-CMV-t-PAGFP Vector. The amplified PCR product of tissue plasminogene activator and construction map of pl-CMV-t-PAGFP vector is given in Fig 3. M is the TaKKara marker and other one is the sample of tissue plasimogene activator which shows the size of 1800 approximately. The construction map of pL-CMV-tPAGFP is also given in Fig 3. In the Fig 3, 1 and 2 are the sample digested while M is the marker. Fig 3, A is PCR amplification of tissue plasimongene activator and B is the digestion result of plasmidExpression of pl-CMV-t_PAGFP vector in CEF and cPGC cellsFor pl-CMV-t-PAGFP plasmid confirmation, chicken embryo fibroblasts (CEF) and chicken primary germ cells (cPGC) were transfected with pl-t_PAGFP vector. Fig 4 shows the expression of green fluorescence protein in chicken embryo fibroblasts (CEF) and chicken primary germ cells (cPGC). In Figure 4, (A, C) are green image of chicken embryo fibroblasts (CEF) and chicken primary germ cells (cPGC) and B and D are bright- photographs corresponding to A and C field Fig 4, (A, C) are green florescence image of embryo fibroblasts (CEF) and chicken primary germ cells (cPGC), (B and D) are bright-field photographs Corresponding to A and C.The PCR product of ovalbumin gene and construction map of pl-2.8OV-t-PAGFP is given in figure 5. In Figure 5 A, 1 and 2 is amplified product of chicken ovalbumin while B is the digestion result of vector with Afl11 enzyme. Fig 5, A is Amplified PCR product of tissue plasminogene activator and B is the construction map of pl-CMV-t-PAGFP plasmid with digestion result. Culture of cells The continuous culture of oviduct epithelial cells from 1, 3 and 6 days showed the following characteristics. The cells digested by the original generation gather agglomerately and beat same as the heart, most cells appeared polygonal; some appeared fusiform and sometime in clusters. There was close contact between cells which belong to structural characteristics of epithelial cells. The cell clusters were attached to the bottom of the plastic dishes and organized into lumen-like structures. The culture results of Oviduct epithelial cells of 1, 3, 6, days are given below in Fig 7 Fig 6, Culture of oviduct epithelial cells, A First day, B third day and C 7 day culture Primary oviduct eptithelial cells appeared spindle, fibroblast-like, satellite and circular shape. They gather agglomerately and beat same as the heart and day vice looked polygonal; some appeared fusiform and sometime in clusters. They appeared in very close with each other and clusters were attached to the bottom of the plastic dishes and organized into lumen-like structures. The culture results of Oviduct epithelial cells of 1, 3, 6, days are given below in Fig 7For pl-2.8OV-t-PAGFP plasmid confirmation, primary oviduct , hela and C127 were transfected with pl-2.8OV-t_PA-GFP and control pEGP-N1 vector oviduct specific vector was only expressed in the oviduct cells but not in hella and C17 cells while control vector was expressed in all cells including oviduct , hela and C127 cells. Fig 6 shows expression of green fluorescence protein in oviduct epithelial cells, hella and C17 cells. Fig 9, GFP expression in oviduct epithelial cells of laying hens, Hella and C17 cellsTransgenic detectingThe lentivirus was injected in 50 eggs out of which 11 (22%) hatched, 4 (36%) out of 11 chicks were detected to contain viral vector sequences by DNA dot blotting analysis. For more confirmation all hatched chicks were also confirmed by PCR suing EFPF specific primers. All four positive chicks were confirmed to carry the GFP as expected in dot blot analysis. Fig 8, A and B shows the PCR and dot blot analysis result of all chicks including non transgenic, genome from oviduct cells transfected with oviduct specific vector and 1-11 transgenic chicken. Fig 8 A, (M), marker (Tiangen) , (C) negative control, genome DNA from non-transgenic chicken, (P) positive control, genome DNA from oviduct cells transfected with oviduct specific promoter chicken and plasmid pL- 2.8OV-tPAGFP (Line 1-11) transgenic chickens. P is the positive control which is the genome of oviduct cells transfected with oviduct specific vector and C is the negative control which is DNA from the non transgenic chicken. Fig 8 B, (C) Negative control, genome DNA from non-transgenic chicken, (P) positive control, genomic DNA from oviduct cells transfected with plasmid pl-2.8OV-Tpagfp 10ng, (1-11) hatched chickens.DiscussionThe results of oviduct epithelial cells cultured in this study are in agreement with results of Gao et al., 2005, Nadia et al, 1989 and Moll et al. 1986 they reported that oviduct epithelial cells look like spindle, in cluster, fusiform and are closely associated, these are the marking characteristic of epithelial cell identity. After 48 hours of transfection, expression scales of pl-2.8OtPAGFP vector were highest in the primary chicken oviduct epithelial cells but not in hella and C17 cells. It shows that oviduct-specific expression vector can only drive exogenous genes specific expression in the oviduct epithelial cell. It has been gradually recognized that DNA transfection with chicken oviduct cells is problematic (Hiroshi et al, 1998). Firstly, it depends on the primary culture, which is not easily transfected due to unavailability of cell line. Secondly, the expression of endogenous chicken ovalbumin gene is strictly limited to the oviduct epith