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ISSN : 1738-2432(Print)
ISSN : 2288-0151(Online)
Reproductive & developmental Biology Vol.37 No.3 pp.91-96
DOI : https://doi.org/10.12749/RDB.2013.37.3.91

Unanticipated Gene Deletion in the Transgenic Chicken Employing Ovalbumin Promoter for Oviduct Specific Expression

Tae Young Jang1, Bon Chul Koo2, Mo Sun Kwon2, Ji Yeol Roh2, Teoan Kim2, Young Sik Park1,†
1Department of Zootechnical Science, KyungPook National University, Daegu 702-701, Korea
2Department of Physiology, Catholic University of Daegu School of Medicine, Daegu 705-718, Korea
(Received: 28 August 2013/ Accepted: 5 September 2013)

Abstract

Transgenic chickens have been spotlighted as an highly potent bioreactor for their fecundity, short generation time,and eggs associated with mass production of protein. In this study, we generated transgenic chickens exhibiting oviductspecific expression of human growth hormone fused to human transferrin for oral administration. Gene of themodified growth hormone located at downstream ovalbumin promoter (∼3.6 kb) was introduced to stage X blastodermalcell employing retrovirus vector system. Several transgenic chickens were successfully generated. However,genomic analyses showed unexpected deletion within the transgene. The modification of the transgene seemed tooccur during germ cell formation because the deletion was detected only from the sperm DNA of the G0 founderanimal. There was no evidence of deletion in the somatic cell DNA samples of the same chicken. Consequently, samepattern of the deletion was confirmed in both somatic and germ cells of the G1 progeny.

01 Tae Young Jang.pdf391.7KB

INTRODUCTION

 Transgenic chickens have been considered as highly potent bioreactors for their fecundity and short genera-tion time (Ivarie, 2003). Various embryonic stages can be easily observed and manipulated by a number of me-thods. Furthermore, compared to other mammals high-er similarity between human and chicken proteins es-pecially in glycosylation patterns has been reported (Ra-ju et al., 2000). Above all things, natural sterile micro-environment of the egg system and inherent protease inhibitors in eggs are believed to provide optimal con-dition as it stabilize the biological activity of foreign proteins (Mozdziak and Petitte, 2004; Rapp et al., 2003). Two methods, retrovirus-mediated gene transfer (Thora-val et al., 1995) and direct DNA transfection (Murama-tsu et al., 1997) are mainly considered to transfer fo- reign genes to blastodermal stem cells at stage X (Eyal- Giladi and Kochav, 1976). Especially, retrovirus vector system has been more preferred for its technical ease and effectiveness of gene transfer. Until now, several su-ccessful generation of transgenic chickens using retro-virus vector has been reported, including human ery- thropoietin (Koo et al., 2010), human interferon α-2b (Rapp et al., 2003), human interferon β-1a (Lillico et al., 2007) as well as monoclonal antibodies (Kamihira et al., 2005). In this study, we performed an experiment using ovalbumin promoter enabling oviduct specific ex-pression of the human modified growth hormone with-in ovalbumin that comprises majority of egg white pro-teins. We carried out vector construction, virus prepa-ration, and injection of concentrated virus stock into stage X embryonic stem cell of eggs. As the result. we could detect two G0 transgenic chickens through PCR reaction. One of them mated with normal hen resulting in production of G1 progenies. Four months later when G1 was matured enough to collect sperm, we took the samples of sperm and blood from G0 and G1 chickens, then tested whether the integrated sequence was well preserved or not. Through several tests, we found that the integrated transgene was shortened mainly by dele-tion of most ovalbumin promoter region. Furthermore, DNA sequencing showed that this phenomenon has no-thing to do with previously known splicing donor/ac-ceptor interaction sites.

MATERIALS AND METHODS

Construction of Retrovirus Vector and Virus Production

 The plasmid pLNOv36-hGH-helical-hTf-W we used in this study was constructed by switching CMV promo-ter of pLNCX retrovirus vector (Miller and Rosman, 1989) with a fragment consisting of chicken ovalbumin promoter, hGH-helical-hTf, and woodchuck hepatitis vi-rus posttranscriptional regulatory element (WPRE) (Fig. 1). The WPRE sequence (GenBank accession number M-11082) was introduced following the strategy of Zuffer-ey et al (1999). About 3.6 kb of ovalbumin promoter fragment was obtained by manipulation of upstream re-gion of chicken ovalbumin gene (Lillico et al., 2007), and a fragment of hGH-helical-hTf was prepared by fusion of human growth hormone gene (651 bp) and human transferrin gene (2037 bp, Genbank accession no. NM_ 001063.3) through short helical sequence (22 bp). Fig. 1 shows schematic representation of pLNOv36-hGH-heli-cal-hTf-W. In constructing retrovirus producing cells, we used our established protocol (Kim, 2002). Briefly, PT67 packaging cells (purchased from Clontech) were tran-siently transfected with pLNOv36-hGH-helical-hTf-W, and LNOv36-hGH-helical-hTf-W viruses harvested from the transfected cells were added to the culture of GP2-293 cells (purchased from Clontech). PT67 cells are retro-virus packaging cells characterized by expression of the Gibbon ape leukemia virus envelope gene and gag and pol genes of the MoMLV (Moloney murine leukemia vi-rus), whereas GP2-293 cells have been designed to ex-press olny the gag and pol genes of the MoMLV. The GP2-293 cells infected with LNOv36-hGH- helical-hTf- W were selected with G418 (600 μg/ml) for 2 weeks and the resultant G418R (or Neomycin-resistant) cells we-re transfected with pVSV-G (purchased from Clontech) to provide vesicular stomatitis virus glycoprotein that can package the retroviral RNA genome. Viruses were harvested 48 hours post-transfection. All cells, including virus-producing cells, were grown at 37℃ in a 5% CO2 atmosphere in Dulbecco’s modified Eagle medium (DM-EM) containing 4.5 g/L of glucose (GibcoBRL, USA)) and supplemented with fetal calf serum (10%), penicillin (100 U/ml), and streptomycin (100 μg/ml). The virus- containing medium harvested from the virus-producing cells was centrifugally concentrated to 1/1,000 of the original volume and filtered through a 0.45 μm po-re-sized filter. The virus titer of the concentrated stock was 1×109  NeoR cfu/ml (neomycin-resistant colony-form-ing unit/ml) on both NIH3T3 cells and primary cultu-res of chicken embryonic fibroblast cells (data not sh-own).

Fig. 1. Structure of the LN-Ov36-hGH-helical-hTf-W provirus. LTR, long terminal repeat; NeoR, Neomycin-resistant gene; Ov36, ~3.6 kb ovalbumin promoter; hGH-helical-hTf, human growth hormone gene fused to human transferrin gene by helically structured linker; WP-RE, woodchuck hepatitis post-transcriptional regulatory element gene. The approximate positions of the probes for Southern blotting and of the PCR primer sets were indicated as arrows with two (probes) or one head (primer), respectively. Enzymatic digestion of the provi-rus with Nhe I separates 9783 base pair fragment. Drawing is not to scale.

 In this experiment, only fertilized eggs weighing am-ong 60 to 65 were used. Selected eggs were incubated for 5 hours prior to virus infection. Meanwhile surro-gate egg shells were prepared by decanting egg whites and yolks through 33 mm in diameter holes made on the edge. To minimize contamination, all egg shells we-re washed with sterile water. Whole egg contents of each pre-incubated fertilized egg were collected in a 100 mm petridish, and flip the yolk upside down us-ing sterilized spoon. Five μl of concentrated virus st-ock in DMEM supplemented with polybrene (10 μg/ ml) was injected into the central part of the blasto-derm. After transferring the injected egg contents into the surrogate shell, the marginal space was filled with egg white before sealing the hole with plastic wrap. The sealed eggs were incubated at 37.5℃ and 60% re- lative humidity with a 90° rocking motion every 15 min for 3 days. After three days of incubation, the em-bryos were transferred to larger empty recipient egg-shells through a 42-mm circular window before second incubation at 37℃ and 70% relative humidity with a rocking motion every 30 min for additional 15 days. At 20 or 20.5 days of incubation, the plastic wrap was re-placed with a 60 mm petridish lid and the eggs were allowed to hatch without rocking motion.

PCR Analysis

 Genomic DNA was extracted from chicken muscle ti-ssue and blood using a genomic DNA purification kit (Promega, USA). For PCR analysis, primer sets were designed for the hGH, hTf, helical linker, WPRE, NeoR genes based on the nucleotide sequence of pLNOv36- hGH-helical-hTf-W. The forward and reverse primer pairs correspond to the pLNOv36-hGH-helical- hTf-W nucleotide sequences of 6304-6658 (for hGH), 8785- 9012 (for hTf), 7939-8215 (for helical linker), 9325-9641 (for WPRE), and 1790-2007 (for NeoR). Each reaction mixture consisting of 1 μg of genomic DNA extract, 50 pmol of each primer, 5 μl 10ⅩPCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP was added with water to bring the total reaction volume to 50 μl. The mixture was heated to 94℃ for 5 min prior to the addition of 2.5 U of Taq polymerase (Promega, USA). The amplification profile consisted of 94℃ for 30s (denaturation), 54℃ for 30s (annealing), and 72℃ for 30s (extension). After 35 amplification cycles, the samples were retained at 72 ℃ for 7 min to ensure complete strand extension. The sequences of each primer sets are listed on Table 1.

Table 1. Primer sequences for PCR analysis

Southern Blot Analysis

 For Southern blot analysis, genomic DNA (12 μg) was digested with Nhe I, and separated on a 0.8% agarose gel. The probe corresponding to NeoR DNA fragment  (729 base pair) was synthesized using the PCR DIG Probe Synthesis kit (Roche, Basel, Switzerland) with the primer set of 5'-AAGCTATTCGGCTATGACTG-3’ (upst-ream) and 5'-AAGAAGGCGATAGAAGGCGA-3’ (down-stream). The resulting probe was labeled with digoxin alkaline phosphatase and purified by agarose gel elec-trophoresis before hybridization. Detection of labeled DNA on the positively charged nylon membrane was per-formed using a DIG luminescent detection kit (Roche, Germany).

DNA Sequencing of Genomic Regions Flanking the Provirus

 Genomic DNA was amplified with PCR using NeoR forward primer and WPRE reverse primer. Amplified DNA was electrophorsed through 1% agrose gel for 15 minutes with 120 voltage. DNA was eluted using Gel Extraction Kit (Dok Do PrepTM ELPIS BIOTECH, Ko-rea). After cloning the extracted DNA fragment into the TA cloning vector kit (RBC Bioscience, Taiwan), the se-quencing was performed by BIONICS CORPORATION (Seoul, Korea).

RESULTS

PCR Analysis of Blood and Sperm of G0 Transgenic Chicken

 After injection of highly concentrated LNOv36-hGH- helical-hTf-W virus into the subgerminal cavity of the chicken blastoderm, 36 of 163 eggs were hatched. Thir-teen females and two males were identified as trans-genic chickens by blood PCR analysis (data not sh-own). Six months later, to assure germline transmissi-on, we tested sperm genomic DNA of two male trans-genic chickens (Chicken numbers 41 and 42). PCR anal-ysis determined only one chicken (chicken #41) being transgenic. Interestingly, however, profile of the PCR bands were different depending on the source of the ge-nomic DNA: PCR amplification of blood DNA showed all bands expected, while all genes except NeoR gene were missing from sperm DNA samples (Fig. 2-A).

Fig. 2. A) PCR analysis of G0 transgenic chicken (chicken #41). Genomic DNA samples were derived from sperm and blood. P, plasmid pLNOv36-hGH-helical-hTf-W; N, Normal chicken geno-mic DNA; S, sperm genomic DNA; B, blood genomic DNA. (B) PCR analysis of muscle genomic DNA of G0 and G1 transgenic chicken.

Muscle Tissue DNA PCR Analysis of G0 and G1 Tr-ansgenic Chickens

 G0 chicken (#41) was mated with normal hen and one of twelve progenies was confirmed to be trans-genic. However, as the G1 was reaching to 6 month old, both G0 and G1 chickendied for unknown rea-sons. PCR analysis of muscle genomic DNA isolated from frozen G0 transgenic chicken carcass showed sa-me profile of bands as shown in the blood sample of same chicken (“B” lane of Fig. 2-A and G0 lane of Fig. 2-B). In case of muscle genomic DNA isolated from frozen G1 transgenic chicken carcass, however, some PCR bands were missing as shown in the sperm sam-ple G0 transgenic chicken (“S” lane of Fig. 2-A and G1 lane of Fig. 2-B). We presume that the central part of transgene which encompasses much of Ov36 promoter and hGH-helical-hTf region was vanished in germline of G0 transgenic chicken.

Estimation of Transferred Gene Length

 We performed Southern blotting to confirm the de-leted region of the transgene in G1 muscle genomic DNA. As previously stated, genomic DNA (12 μg) was di-gested with Nhe I and then separated on a 0.8% aga-rose gel before applying NeoR probe. Compared with P lane (positive control of diluted plasmid DNA) G1 lane shows a ~3.7 kb band, indicating deletion of around 6 kb(Fig. 3). We presumed that the gene deletion occu-rred over the regions of Ov36 promoter and hGH-heli-cal-hTf.

Fig. 3. Southern blot analysis of muscle genomic DNA isolated from G1 transgenic chicken. Three different DNA samples (P, plasmid DNA; N, Normal; G1, Transgenic G1 chicken) were di-gested with enzyme Nhe I. NeoR probe was used to detect the bands.

Sequencing of the Transgene Integrated into G1 Muscle Genomic DNA

 We conducted PCR amplification of muscle genomic DNA extracted from the muscle of G1 transgenic chick-en using  NeoR (Forward) and WPRE (Reverse) primer set. The resulting PCR amplified fragment of around 1.4 kb was sequenced by BIONICS CORPORATION (Seoul, Korea) (Fig. 4-A). It has been known that splicesome recognize two dinucleotide sites; GT and AG as spli- cing donor and splicing acceptor, respectively. However, the sequencing data showed two separate splicing ev-ents: One is between GT and TG, and the other be-tween GC and AA. It was estimated that, deletion of as many as 6,022 bp occurred as summarized in Fig. 4-B.

Fig. 4. (A) DNA sequencing data. Putative splicing donor and acceptor sites are marked in bold. (B) Schematic deleted region of LNOv36- hGH-helical-hTf-W provirus. The presumable deletion regions were shown in gray.

DISCUSSION

 In this study, we aimed to generate transgenic chick-ens using retrovirus vector which is composed of es-sential elements for efficient retrovirus production and ovalbumin promoter enabling oviduct specific express-ion of the human growth hormone gene modified by linking of the 3’ end to the human transferrin gene via helical linker sequence (Amet et al., 2009). Inserted hTf gene facilitates oral delivery of hGH-helical-hTf fusion protein (Amet et al., 2010). Thirty six eggs were hatch-ed from 163 eggs undergone retrovirus-mediated gene tansfer. Among the progenies, 15 chickens were veri-fied as transgenic chickens through PCR analysis of bl-ood genomic DNA (13 females and 2 males). Sperm of two transgenic roosters (chicken numbers 41 and 42) we-re collected to perform genomic DNA PCR analysis. As the result, chicken #42 proved absence of transgene in the germ cell, while chicken #41 showed massive dele-tion within the transgene. Afterwards, we conducted muscle genomic DNA PCR analysis of G1 transgenic hen (offspring of chicken #41) then found same dele-tion as observed in #41 sperm genomic DNA gene. Th-rough the Southern blotting of G1 muscle genomic DNA, we could estimate the length of truncated sequence be-ing approximately 6 kb. Finally, we also tasked DNA sequencing to identify deletion of 6,022 bp. One inter-esting point to be considered is that deletion was ob-served only in the DNA isolated from sperm. No dele-tion was detected from the DNA of muscle cells of G0 founder chicken. Taken together, the most plausible ex-planation for the unexpected deletion might be due to unique features of germline cells, such as rapid pro-liferation, etc. It has been well known that the expre-ssion of partly deleted viral genome through recombi-nation is superior to the expression of viral genome re-taining whole sequence (Parr et al., 2009). The signifi-cance of this study may stems from that this is the fir-st report on the retrovirus-mediated gene transfer sys-tem in which deletion of the transferred transgene oc-curs only in the germ cells, although exact mechanism of this phenomenon is yet to be identified. Considering that retrovirus vector has been regarded as one of the best gene transfer system in terms of transgene stabili-ty, more studies of the aberrant gene deletion mecha-nism must be done for future application of retrovirus vector system to the transgenic animal production.

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