Cell Tissue Res (2006) 324: 41–53DOI 10.1007/s00441-005-0060-9

REGULAR ARTICLE

Paul E. Mozdziak . Qian Wu . Jennifer M. Bradford .Samuel L. Pardue . Suparerk Borwornpinyo .Carol Giamario . James N. Petitte

Identification of the lacZ insertion site and beta-galactosidaseexpression in transgenic chickens

Received: 9 May 2005 / Accepted: 25 July 2005 / Published online: 12 January 2006# Springer-Verlag 2006

Abstract The quail:chick chimera system is a classicalresearch model in developmental biology. An improve-ment over the quail:chick chimera system would be a lineof transgenic chickens expressing a reporter gene. Trans-genic chickens carrying lacZ and expressing bacterialbeta-galactosidase have been generated, but complete char-acterization of the insertion event and characterization ofbeta-galactosidase expression have not previously beenavailable. The genomic sequences flanking the retroviralinsertion site have now been identified by using inversepolymerase chain reaction (PCR), homozygous individualshave been identified by using PCR-based genotyping, andbeta-galactosidase expression has been evaluated by usingWestern analysis and histochemistry. Based upon the cur-rent draft of the chicken genome, the viral insertion carryingthe lacZ gene has been located on chromosome 11 withinthe predicted gene for neurotactin/fractalkine (CX3CL1);neurotactin mRNA expression appears to be missing fromthe brain of homozygous individuals. When Generation2 (G2) lacZ-positive individuals were inter-mated, theygenerated 361 G3 progeny; 82 were homozyous for lacZ(22.7%), 97 were wild-type non-transgenic (26.9%), and182 (50.4%) were hemizygous for lacZ. Western analysisrevealed the highest expression in the muscle and liver.With the identification of homozygous birds, the line ofchickens is now designated NCSU-Blue1.

Keywords Beta-galactosidase . Neurotactin .Inverse polymerase chain reaction . Genotyping .lacZ . NCSU-Blue1 . Chicken

Introduction

Transgenic technology is a useful tool amongst the rep-ertoire of experimental approaches available to cell bi-ologists. In many model organisms, such as Drosophila,Caenorhabditis elegans,Xenopus, zebrafish, and themouse,the insertion of genes into the germline has become astandard method to study gene function or to trace cellsduring development. Whereas the avian embryo has beena model vertebrate organism for more than a century, theproduction of transgenic chickens as models to gain knowl-edge concerning embryonic development has been rare.Although embryonic transgenic somatic chimeras havebeen employed in some studies (Epstein et al. 1994; Ishiiet al. 2004), avian transgenic systems have seldom beeninvestigated in detail, because the driving force behindavian transgenic technology has been the promise of thecheap production of therapeutic proteins with the domes-tic hen as a bioreactor (Sang 1994; Harvey and Ivarie2003; Ivarie 2003; Rapp et al. 2003). Recent attempts toproduce transgenic chickens have been successful at ex-pressing bacterial beta-lactamase in the egg white as proof-of-principle for the production of pharmaceutical proteins ineggs (Harvey et al. 2002a,b; Harvey and Ivarie 2003).Furthermore, biologically active human interferon alpha-2bis deposited in the eggs of transgenic hens (Rapp et al.2003). However, few investigators have sought to generatetransgenic chickens as an improved system for cell lineageanalysis over the classic quail-chick chimera system. Onetransgenic approach for cell lineage analysis is to producechickens with stably integrated bacterial lacZ expressingbeta-galactosidase. The endeavor to create these chickenshas been highly challenging, and many investigators havebeen unsuccessful at generating transgenic chickens ex-pressing this enzyme (Vick et al. 1993; Thoraval et al. 1995;Harvey et al. 2002a).

This work was supported by the College of Agriculture and LifeSciences of North Carolina State University under projectnos. 06590 (P.E.M.) and 01868 (J.N.P.) and in part by NationalResearch Initiative Competitive Grant no. 2005-35206-15241 fromthe USDA Cooperative State Research, Education, and ExtensionService (P.E.M.).

P. E. Mozdziak (*) . Q. Wu . J. M. Bradford . S. L. Pardue .S. Borwornpinyo . C. Giamario . J. N. PetitteDepartment of Poultry Science,North Carolina State University,Raleigh, NC 27695, USAe-mail: pemozdzi@unity.ncsu.eduTel.: +1-919-5155544Fax: +1-919-5152625

The major challenge in transgenic chicken productionis that a fertile, freshly laid, chicken egg contains an em-bryo with approximately 50,000 to 60,000 cells (Spratt andHaas 1960) at the time that the chicken egg is most ac-cessible for manipulation, whereas in a mammalian system,the investigator only has to deliver the gene of interest to asingle cell. Love et al. (1994) and Ono et al. (1994) havebeen able to manipulate the newly fertilized chick or quailegg, but a hen must be killed for each manipulation, mak-ing it inconvenient and costly to perform the necessaryexperiments. Although some success has been achievedin generating transgenic chickens by using microinjectionof DNA into the fertilized egg (Love et al. 1994), thegreatest advances have occurred through employing fresh-ly laid eggs in combination with replication-incompetentretroviral or lentiviral gene delivery systems (Bosselmanet al. 1989a,b; Mozdziak et al. 2003a; Rapp et al. 2003;McGrew et al. 2004; Kwon et al. 2004).

The first successful transgenic chicken was produced byusing a replication-incompetent retroviral vector in 1989when Bosselman et al. (1989a) demonstrated germlinetransmission of neomycin (Tn5) and HSV-1 thymidine ki-nase after a replication-incompetent spleen necrosis viral(SNV) vector was introduced into early embryos. Trans-genic birds carrying the lacZ gene were also produced byusing a replication-incompetent avian leukosis retroviralvector (Thoraval et al. 1995). Unfortunately, beta-galacto-sidase expression was only noted in cultures of embryonicfibroblasts, and expression was not reported in the entireembryo (Thoraval et al. 1995). Mizuarai et al. (2001) pro-duced transgenic quail carrying green fluorescent protein(GFP) as a reporter gene, but the embryos and birds failedto express any protein.

Although other investigators have recently producedchickens expressing reporter genes (McGrew et al. 2004;Kwon et al. 2004; Chapman et al. 2005), Mozdziak et al.(2003a) produced the first transgenic chickens specificallyas a resource for cell lineage analysis. Initial character-ization of the transgene demonstrated typical Mendelianinheritance, with nuclear-localized beta-galactosidase ex-pression in the early chick embryo and in cultured satel-lite cells. Subsequently, the transgenic birds were used ina study showing that the intestinal mucosa could hydrolyzelactose, thereby verifying the functional activity of theenzyme (Mozdziak et al. 2003b). Chicks do not normallycontain the enzymes needed to hydrolyze lactose into use-ful energy sources (glucose and galactose), but the dem-onstrated presence of active beta-galactosidase may make itpossible for the transgenic birds to utilize lactose. The ob-jective of the current study has been the further charac-terization of the lacZ transgenic birds and the establishmentof a homozygous breeding population that will providetransgenic embryos.

Materials and methods

Transgenic chickens

The experimental procedures for the generation of thetransgenic chickens employed in this study were as pre-viously described (Mozdziak et al. 2003a,b). The birdswere developed by using an SNV vector encoding nuclear-localized lacZ (Mikawa et al. 1991, 1992; Epstein et al.1994) driven by the viral long-terminal repeat (LTR).Previously, the insertion of the lacZ gene was confirmed byusing the polymerase chain reaction (PCR) and followedexpected Mendelian inheritance. Expression of active beta-galactosidase in situ was demonstrated in the embryo andcultured skeletal muscle cells using X-gal as a substrate.Progeny testing resulted in a single G0 male that trans-ferred the lacZ gene to two Generation 1 (G1) male founderbirds identified as Precious 1 (P1) and Precious 2 (P2).

Southern analysis

The next experiment beyond those reported in Mozdziaket al. (2003a,b) was to confirm the integration of thelacZ gene into the genome by using Southern analysis.The presence of the lacZ gene was determined by digest-ing genomic DNA from the two founder animals (P1, P2)and DNA from their G2 offspring that carried the lacZgene. Blood was harvested from the animals, and DNAwas isolated by using a modification of the protocol ofPetitte et al. (1994). Briefly, blood was diluted 1:10 withphosphate-buffered saline (PBS), mixed with lysis buffer(10 mM TRIS HCl pH 7.5, 5 mM MgCl2, 0.32 M su-crose, 1% Triton X-100), and spun at 10,000g for 15 s,and the supernatant was placed in a fresh tube. Sodium-dodecyl sulfate and proteinase-K was added, and proteinswere digested overnight at 37°C with constant rotation.Subsequently, the protein was precipitated by using satu-rated NaCl, and the DNA was precipitated by using eth-anol. All DNA samples were resuspended in TRIS-EDTAbuffer. For Southern analysis, the DNA was digested withKpn1, which cuts DNA in the 5′ viral LTR, with EcoRV,which cuts within the lacZ sequence, and with HinDIII,which excises lacZ (Fig. 1). Subsequently, 20 μg of eachdigested DNA sample was fractionated through a 1%agarose gel, denatured, and transferred to a nylon mem-brane. The membrane was hybridized with the 3-kb lacZfragment generated from a BamHI digestion of pMiwZ(Kadokawa et al. 1990; Ono et al. 1994). The lacZ-spe-cific sequence was radiolabeled with 32P dCTP, and theblots were exposed to Kodak AR X-ray film. The labelingof the lacZ probe and Southern hybridization were per-formed by Lark Technologies (Houston, Tex.).

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Isolation, cloning, and sequencingof the viral insertion

The genomic region flanking the SNTZ provirus was am-plified by inverse-PCR (I-PCR) following a protocol mod-ified from Kim et al. (2003). Approximately 4 μg DNAfrom the blood of transgenic chicken P1 was digested with90 U XbaI (Promega, Madison, Wis.) for 2 days in a totalvolume of 60 μl. After inactivation of the enzyme, theDNA was circularized by ligation with 15 U T4 DNA li-gase (Promega) in a total volume of 400 μl at 16°Covernight. Circular DNAwas precipitated with ethanol andresuspended in 30 μl TRIS-EDTA buffer. PCR was per-formed with 1 μl DNA template, 0.2 mM dNTPs (TaKaRaBio, Japan), 10 pmol each primer, PCR buffer (TaKaRaBio), and 2 U Taq polymerase. The primers were designedto amplify the region preceeding the XbaI site in the LTRregion of the SNTZ vector. The primers were 5′-ATCTCCTCTCACTGCCAATC-3′ and 5′-GGTGATACTAAGTCGTGGC-3′. The DNA was amplified by using 30cycles of 94°C for 10 s, 56°C for 30 s, and 68°C for 6 min.Amplification products were cloned into the AccepTorvector (Novagen, EMD Biosciences, Madison, Wis.). TheDNA was sequenced at the DNA Sequencing Facility ofIowa State University. Sequence data were compared withthe chicken genome database available at http://www.ensembl.org.

After the five prime flanking sequence had been iden-tified, the three prime flanking sequence was amplified byemploying a lacZ forward primer (5′-TTC TGTATG AACGGT CTG GTCC-3′) and a reverse primer (5′-TCC ACAGCA CAA GCA CTA TTC-3′) based upon the predictedgenomic integration site revealed by using the location ofthe sequence 5′ to the proviral insert. The sequence wasamplified by Taq polymerase with an initial denaturation at95°C for 3 min followed by 40 cycles of 95°C for 30 s,58°C for 30 s, 70°C for 4 min, and one final extension of70°C for 10 min. The PCR fragment was fractionatedthrough an agarose gel, extracted from the gel, and ligated

into pGEM-T Easy vector (Promega). Subsequently, theplasmid DNA containing the amplicon was sequenced atthe DNA Sequencing Facility of Iowa State University. Thesequencing results were further analyzed by using BLAST(Altschul et al. 1990) and Bioedit (Hall 1999) to comparethe PCR results between P1, P2, neurotactin, and theavailable sequence for SNTZ.

Genotyping of G3 transgenic chicken

Multiplex PCR with three different primers was employedto identify homozygous and hemizygous individuals. Twoprimers were designed based on the predicted chickengenomic sequence. The first primer was upstream of thepredicted proviral insert (5′-CACGCACTAACATCCAATTCCC-3′), the second primer was downstream of the lacZinsert (5′-TCCACAGCACAAGCACTATTCC-3′), and thethird primer was a lacZ-specific reverse primer (5′-CCTCTTCGCTATTACGCCAG-3′). DNA samples were ex-tracted from the blood of G3 transgenic chicks. Multiplex-PCR was performed in 20 μl with 0.2 mM dNTPs (TaKaRaBio), 10 pmol each of the three primers, PCR buffer(TaKaRa Bio), MasterAmp PCR Enhancer (Epicentre,Madison, Wis.), and 1.5 U Taq polymerase. The DNAwasamplified at 94°C for 3 min, followed by 30 cycles of94°C for 30 s, 58°C for 30 s, and 70°C for 2.5 min, and afinal extension at 70°C for 10 min. The predicted size ofthe amplification products for a non-transgenic wild-typebird was a single 522-bp band specific to chicken geno-mic DNA. In the event of a transgenic animal, the ge-nomic-specific primers would be specific to a fragmentgreater than 4 kb, which would not amplify under thePCR conditions employed in this study. A hemizygouschick was predicted to produce two PCR fragments. Thefirst PCR fragment generated by a hemizygote waspredicted to be approximately 1,200 bp from the lacZ-specific reverse primer indicative of a transgenic allele.The second fragment was predicted to be 522 bp indic-

Fig. 1 a Restriction map of SNTZ inserted into the transgenicchicken genome (LTR long-terminal repeats of the spleen necrosisvirus; SNV). The restriction enzyme KpnI cuts the SNV LTR,EcoRV cuts once within the lacZ sequence, and HinDIII exciseslacZ (probe lacZ probe generated through BamHI digestion of the

plasmid pMiwZ and employed in the Southern analysis). b Southernblot of DNA from Precious 1 (P1), Precious 2 (P2), and their secondgeneration progeny (G2). DNAwas digested with KpnI, EcoRV, andHinDIII

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ative of a non-transgenic allele. A homozygous chick waspredicted to produce only a 1,200-bp fragment indicativeof two transgenic alleles.

Reverse transcription/PCR for neurotactin

RNA was isolated from the brains of lacZ homozygous,lacZ heterozygous, and wild-type chickens by using Trizol(Invitrogen, Carlsbad, Calif.). Brain was chosen as the testtissue because neurotactin has been proposed to be highlyexpressed in neurons (Cook et al. 2001). cDNA was re-verse-transcribed with M-MLV reverse transcriptase. DNAwas amplified by Taq polymerase after a 3-min 95°Cdenaturation step using 35 cycles of 95°C for 30 s, 58°C for30 s, and 68°C for 6 min, followed by a 10-min finalextension at 68°C. A forward primer lying within thepredicted sequence for exon 1 (5′-TAG GCA GCC AGGAGT GAA AC-3′), a forward primer lying within the pre-dicted sequence for exon 2 (5′-ACC TCT GAA GTG TTCAAA ATG G-3′), and a common reverse primer lying withthe predicted sequence of exon 3 (5′-GTG AAG GTG GTACTT GGA GAC-3′) was employed in the PCRs. Theintegrity of the RNA was confirmed by amplifying allsamples with primers specific for D-glyceraldehyde-3-phosphate dehydrogenase (5′-TGA CCT TTC TCT CCTCTCTCC-3′; 5′-AAGCACCAGCAATCCTTT TCC-3′).

Expression analysis

Beta-galactosidase expression was evaluated by isolatingthe protein from heart, lung, gizzard, small intestine, liver,and brain of 28-week-old G3 transgenic chickens. Fur-thermore, skeletal muscle was harvested from five agedG2 chickens (>2 years of age) to evaluate expressionacross individuals. Protein expression was only evaluatedfrom P1 progeny because the Southern analysis and theanalysis of the insertion site showed that the location ofthe lacZ transgene was identical in P1 and P2. Protein wasisolated by homogenizing each tissue with lysis buffer(20 mM TRIS-HCl, 10 mM NaCl, 10 mM KCl, 3 mMMgCl2, 1 mg/ml phenylmethane sulfonylfluoride). Proteinconcentration was determined for each homogenate by theBicinchonic Acid Assay (Sigma, St. Louis, Mo.). Sub-sequently, each homogenate was mixed 1:1 with samplebuffer (62.5 mM TRIS-HCl pH 6.8, 2% SDS, 25% glyc-erol, 0.01% Bromophenol Blue; Biorad).

The stacking gels were composed of 5% acrylamide0.1 M TRIS-HCl (pH 6.8), 0.1% SDS, 0.1% ammoniumpersulfate, and 0.003% N, N, N′, N′_tetramethylenedi-amine. The separating gels were composed of 8% totalacrylamide (29% acrylamide and 1% N, N′_methylene-bisacrylamide), 0.375 M TRIS (pH 8.8), 0.1% SDS, 0.1%ammonium persulfate, and 0.006% N, N, N′, N′_tetra-methylenediamine. The running buffer consisted of 0.1 MTRIS (base), 150 mM glycine, 0.1% SDS.

Immediately following fractionation of 20 μg proteinthrough each SDS-polyacrylamide gel, the proteins weretransfered to a polyvinylidene fluoride membrane (GEOsmonics, Fairfield, Conn.) in 0.3 M TRIS, 0.8 M glycine,18% methanol for 1 h at 400 mA constant current. Aftertransfer, the membranes were hydrated and blocked for 1 hwith 3% bovine serum albumen in TBST (50 mM TRIS,200 mM NaCl, 0.05% Tween-20 pH 7.5). Subsequently,the primary anti-beta-galactosidase monoclonal antibody(JIE7, Developmental Studies Hybridoma Bank, IowaCity, Iowa) was diluted 1:1 with the blocking solution andincubated with the blot for 1.5 h at room temperature. Theblot was washed three times with TBST and incubatedwith goat-anti-mouse IgG conjugated to horseradish per-oxidase diluted 1:2,500 in blocking solution for 1.5 h. Thesecondary antibody was detected with the Immuno-Stain-HRP Chemi-luminescent Kit (Bio-Rad) and X-ray film.

Tissue was fixed in 4% paraformaldehyde in PBS,infiltrated with 20% sucrose, and frozen in isopentane.Section (10 μm thick) were cut on a cryostat and incubatedin X-Gal solution (1 mg/mL X-Gal PBS pH 7.4, 5 mMpotassium ferrocyanide, 5 mM potassium ferricyanide,2 mM MgCl2, 0.2% Triton X-100) overnight in the darkat 37°C.

Analysis of cell fate

Myogenic satellite cells were isolated from the Pectoralisthoracicus of post-hatch G3 chickens by established pro-cedures (Mozdziak et al. 1996, 2003a), and the satellitecells were maintained in 84% Dulbecco’s Modified Eagle’smedium, 15% fetal bovine serum, and 1% penicillin-strep-tomycin. Briefly, approximately 25,000 satellite cells wereimplanted into the hearts of Stage 14 (Hamburger andHamilton 1951) embryos. The embryos were cultureduntil embryonic day 7–14 by using standard procedures(Giamario et al. 2003), fixed at 4°C with 2% formalde-hyde in PBS pH 7.4 for 30 min, rinsed in PBS, andincubated in X-Gal solution (1 mg/mL X-Gal PBS pH7.4, 5 mM potassium ferrocyanide, 5 mM potassium fer-ricyanide, 2 mM MgCl2, 0.2% Triton X-100) overnight inthe dark at 37°C. Subsequently, the embryos were washedin PBS and stored in 70% ethanol before microscopicevaluation. Similarly, DNA was extracted from the heartsof the embryonic day 14 embryos following proceduresmodified from Mozdziak et al. (2003a). The presence ofthe lacZ gene in such implanted hearts was determined byPCR. Briefly, Taq polymerase was used to amplify a 588-bp fragment of lacZ with the forward primer 5′-TCTGTATGAACGGTCTGGTC-3′ and the reverse primer 5′-ACTTACGCCAATGTCGTTATC-3′. The DNA was ampli-fied by using 35 cycles of 95°C for 30 s, 54°C for 1 min,and 72°C for 1 min in a thermocycler (PTC-200, MJResearch, Waltham, Mass.). Subsequently, the amplifica-tion products were fractionated through a 1.5% agarosegel to reveal the presence of the 588-bp lacZ fragment.

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Avian Transgenic

P1 TAGACACCAG AAAGCCTTCT GATGCTCCCC CAACCGCCCT CAGAANTGAC CAAAAGCCAT

P2 ---------- ---------- ---------- ---------- ---------- ----------neurotactin ----CACCAG AAAGCCT~CT GATGCTCCCC CAACCGCCCT CAGAAATGAC ~AAAAGCCATSNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 CAGCAGGGCT GCACGGCTTC CTTGCAGCCA GGCTGTTTGC TCCCCGACAG CTTCCTACCCP2 ---------- ---------- ---------- ---------- ---------- ----------neurotactin CAGCAGGGCT GCACGGCTTC CTTGCAGCCA GGCTGTTTGC TCCCCGACAG CTTCCTACCCSNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 AGCCTGTTCC TGCCGCAGGA TTTCCCAAGC CAAGGAAGGC GTGAGGTCCC TTCATGCCTGP2 ---------- ---------- ---------- ---------- ---------- ----------neurotactin AGCCTGTTCC TGCCGCAGGA TTTCCCAAGC CAAGGAAGGC ATGAGGTCCC TTCATGCCTGSNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 TAACCTGATC CAGAGCTGAC TTCCTGCTGT GTACAATCCC CTGGCCCCAC GCACTAACATP2 ---------- ---------- ---------- ---------- ---------- ----------neurotactin TAACCTGATC CAGAGCTGAC TTCCTGCTGT GTACAATCCC CTGGCCCCAC GCACTAACATSNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 CCAATTCCCC AGTCCCTTGC ATAAAGTCAT AATCCCTCAC GTGGAGCCAC AGATCCCTCCP2 ---------- ---------- ------TCAT AATCCCTCAC GTGGAGCCAC AGATCCCTCCneurotactin CCAATTCCCC AGTCCCTTGC ATAAAGTCAT AATCCCTCAC GTGGAGCCAC AGATCCCTCCSNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 GTGAAACCGC AGACCCCTGC ATGAAGCCAT AAACCCCTTT ATGAAGCCGC AGACCCCTGCP2 GTGAAACCGC AGACCCCTGC ATGAAGCCAT AAACCCCTTT ATGAAGCCGC AGACCCCTGCneurotactin GTGAAACCGC AGACCCCTGC ATGAAGCCAT AAACCCCTTT ATGAAGCCGC AGACCCCTGCSNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 ATGAAGCCAT AAACCCCTTT ATGAAGCCAC AGACCCCTCC ATGACACCAC AGTGTGGGAGP2 ATGAAGCCAT AAACCCCTTT ATGAAGCCAC AGACCCCTCC ATGACACCAC AGTGTGGGAGneurotactin ATGAAGCCAT AAACCCCTTT ATGAAGCCAC AGACCCCTCC ATGACACCAC AGT-------SNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 GGAGCTCTGG GGGGAATAGT GCTGGCTCGC TAACTGCTGT ATTAGCTTCT GTACCCATGCP2 GGAGCTCTGG GGGGAATAGT GCTGGCTCGC TAACTGCTAT ATTAGCTTCT GTACCCATGCneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ --------GG GGGGAATAGT GCTGGCTCGC TAACTGCTAT ATTAGCTTCT GTACCCATGClacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 CCGCCATTGT ACTTGATATG CCATTTCTCG GAATCGGCAT CAAGTCTCGC TTCTCGGAATP2 CCGCCATTGT ACTTGATATG CCATTTCTCG GAATCGGCAT CAAGTCTCGC TTCTCGGAATneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ CCGCCATTGT ACTTGATATG CCATTTCTCG GAATCGGCAT CAAGTCTCGC TTCTCGGAATlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 CGGCATCAAG TTTCGCTTCT CGAAATCGGC GTCATTTCTC GGCATCGAGA GCAAGCTCATP2 CGGCATCAAG TTTCGCTTCT CGAAATCGGC GTCATTTCTC GGCATCGAGA GCAAGCTCATneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ CGGCATCAAG TTTCGCTTCT CGAAATCGGC GTCATTTCTC GGCATCGAGA GCAAGCTCATlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 AAACCATAAA AGGAAATGTG TATTGAAGGC AAGCATCAGA CCACTTGCGC CATCCAATCAP2 AAACCATAAA AGGAAATGTG TATTGAAGGC AAGCATCAGA CCACTTGCGC CATCCAATCAneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ AAACCATAAA AGGAAATGTG TATTGAAGGC AAGCATCAGA CCACTTGCGC CATCCAATCAlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 CGAACGAACA CGAGATCGGA CTATCATACT GAGCCAATGG TTGTAAAGGG CAGATGCTACP2 CGAACGAACA CGAGATCGGA CTATCATACT GAGCCAATGG TTGTAAAGGG CAGATGCTACneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ CGAACGAACA CGAGATCGGA CTATCATACT GAGCCAATGG TTGTAAAGGG CAGATGCTAClacZ ---------- ---------- ---------- ---------- ---------- ----------

Fig. 2 a Pairwise alignment ofthe 5′ insertion site of SNTZ inPrecious 1 (P1) and Precious 2(P2). The 5′ insertion site wasdetermined by I-PCR. The P1and P2 sequences are alignedwith chicken neurotactin, theoriginal SNTZ vector, and lacZ(dashes gaps within an align-ment, yellow homologoussequence with predicted neuro-tactin, turquoise homologoussequence with SNTZ, pinkhomologous sequence withlacZ, bright green sequenceof approximately 180 bp pro-posed to contain the nuclearlocalization signal

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P1 TCTCCAATGA GGGAAAATGT CATGTAACAC CCTGTAAGCT GTAAGCGGCT ATATAAGCCG P2 TCTCCAATGA GGGAAAATGT CATGTAACAC CCTGTAAGCT GTAAGCGGCT ATATAAGCCG neurotactin ---------- ---------- ---------- ---------- ---------- ---------- SNTZ TCTCCAATGA GGGAAAATGT CATGTAACAC CCTGTAAGCT GTAAGCGGCT ATATAAGCCG lacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 GGTACATCTC TTGCTCGGGG TCGCCGTCCT ACACATTGTT GTTGTGACGT GCGGCCCAGA P2 GGTACATCTC TTGCTCGGGG TCGCCGTCCT ACACATTGTT GTTGTGACGT GCGGCCCAGA neurotactin ---------- ---------- ---------- ---------- ---------- ---------- SNTZ GGTACATCTC TTGCTCGGGG TCGCCGTCCT ACACATTGTT GTTGTGACGT GCGGCCCAGA lacZ ---------- ---------- ---------- ---------- ---------- ----------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ P1 TTCGAATCTG TAATAAAACT TTTTTTTTTC TGAATCCTCA GATTGGCAGT GAGAGGAGATP2 TTCGAATCTG TAATAAAACT TTTTTTTTTC TGAATCCTCA GATTGGCAGT GAGAGGAGATneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ TTCGAATCTG TAATAAAACT TTTTTTTTTC TGAATCCTCA GATTGGCAGT GAGAGGAGATlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 TTTGTTCGTG GtGTTGGCTC GCCTACTGGG TGGGCGCAGG GATCCGGACT GAATCCGTAGneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ TTTGTTCGTG GTGTTGGCTC GCCTACTGGG TGGGCGCAGG GATCCGGACT GAATCCGTAGlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 TACTTCGGTA CAACATTTGG GGGCTCGTCC GGGATACCCT CCCCATCGGC AGAGGTACCAneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ TACTTCGGTA CAACATT~~~ GGGCTCGTC~ ~GGATACC~~ TCCCATCGGC AGAGGTGCCAlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 ACTGCTTCTT CGAAC~TTTC TTCGAACTCC GGCGCCGGTG AGTAAGTACT TGATTTTGGTneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ ACTGCTTCTT CGAACGCTTC TTCGAACTCC GGC~~~~~~~ ~~~AAGTACT TGATTTTGGTlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 ACCTCGCGAG GGTTTGGGAG GATCGGAGTG GCGGGACGCT GCCGGGAAGC TCCACCTCCGneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ ACCTCGCGAG GGTTTGGGAG GATCGGAGTG GCGGGACGCT GCCGGGAAGC TCCACCTCCGlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 CTCAGCAGGG GACGCCCTGG TCTGT~GGTA TCTGATTGTT GTTGAGCCGT CCCTAAGACGneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ CTCAGCAGGG GACGCCCTGG TCTGTTGGTA TCTGATTGTT GTTGAGCCGT CCCTAAGACGlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 GTGATACTAA GTCGTGGCTT GTGTGTTTGT TTGTTGCCTT GTGTTTGTTC GTCGTTTGTAneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ GTGATACTAA GTCGTGGCTT GTGTGTTTGT TTGTTGCCTT GTGTTTGTTC GTCGTTTGTAlacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 ATTCGAGCTC GCCCGGGGAT CCTCTAGAGG ATCTTCCCGG CCTCTGAGCT ATTCCAGAAGneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ ATTCGAGCTC GCCCGGGGAT CCTCTAGAGA A--------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTTTGCAA AGATGGATAAneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- ---------- ---------- ---------- P1 ---------- ---------- ---------- ---------- ---------- ----------P2 AGTTTTCCGG AATTCCGCAA AAAAGAAGAG AAAGGTAGAA GACCCCAAGG ACTTTCCTTCneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ ---------- ---------- ---------- ---------- ---------- ----------

Fig. 2 continued

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Results

Southern analysis

We had previously demonstrated, by PCR, that the lacZgene was present in the blood of G1 and G2 transgenicchickens, and that the gene was transferred following thepredicted Mendelian ratio (approximately 50% G1 to 50%G2; Mozdziak et al. 2003a). All chickens originated from

one G0 male who produced two G1 progeny: P1 and P2carrying the lacZ gene. P1 and P2 were mated with wild-type female chickens to produce G2 progeny.

Here, the PCR information was extended to Southernanalysis to show that the gene was integrated into thegenome of the transgenic chickens, and that it passed fromthe parent to the offspring. Southern analysis after restric-tion digestion with KpnI showed one large band consistentwith the predicated single junction fragment generated

Avian Transgenic

lacZ ---------- ---------- ---------- ---------- ---------- ----------

P1 ---------- ---------- ---------- ---------- ---------- ----------P2 AGAATTGCTA AGTTTTTTGA GTCCAAGCTT GGCACTGGCC GTCGTTTTAC AACGTCGTGAneurotactin ---------- ---------- ---------- ---------- ---------- ----------SNTZ ---------- ---------- ---------- ---------- ---------- ----------lacZ ---------- ---------- ---------- --CACTGGCC GTCGTTTTAC AACGTCGTGA

P1 ---------- ---------- ---------- ---------- -- P2 CTGGGAAAAC CCTGGCGTTA CCCAACTTAA TCGCCTGCAG CA neurotactin ---------- ---------- ---------- ---------- -- SNTZ ---------- ---------- ---------- ---------- -- lacZ CTGGGAAAAC CCTGGCGTTA CCCAACTTAA TCGCCTGCAG CA

Fig. 2 continued

Avian Transgenic

P1 GAGATCGGAC TATCATTGTG AGCCAATGGT TGTAAAGGGC AGATGCTACT CTCCAATGAGSNTZ GAGATCGGAC TATCATACTG AGCCAATGGT TGTAAAGGGC AGATGCTACT CTCCAATGAGneurotactin ---------- ---------- ---------- ---------- ---------- ----------

P1 GGAAAATGTC ATGTAACACC CTGTAAGCTG TAAGCGGCTA TATAAGCCGG GTACATCTCTSNTZ GGAAAATGTC ATGTAACACC CTGTAAGCTG TAAGCGGCTA TATAAGCCGG GTACATCTCTneurotactin ---------- ---------- ---------- ---------- ---------- ----------

P1 TGGTTGGGGT CGCCGTCCTA CACATTGTTG TTGTGACGtG CGGCCCAGAT TCGAATCTGTSNTZ TGCTCGGGGT CGCCGTCCTA CACATTGTTG TTGTGACGTG CGGCCCAGAT TCGAATCTGTneurotactin ---------- ---------- ---------- ---------- ---------- ----------

P1 AATAAAACTT TTTTTTTTCT GAATCCTCAG ATTGGCAGTG AGAGGAGATT TTGTTCGTGGSNTZ AATAAAACTT TTTTTTTTCT GAATCCTCAG ATTGGCAGTG AGAGGAGATT TTGTTCGTGGneurotactin ---------- ---------- ---------- ---------- ---------- ----------

P1 TGTTGGCTCG CCTACTGGGT GGGCGCAGGG ATCCGGACTG AATCCGTAGT ACTTCGGTACSNTZ TGTTGGCTCG CCTACTGGGT GGGCGCAGGG ATCCGGACTG AATCCGTAGT ACTTCGGTACneurotactin ---------- ---------- ---------- ---------- ---------- ----------

P1 AACACACAGA CCCCTCTGTG AAGCCACAGA CCCCTCCCAG CTCTCGTGGG GAGCCCAAGGSNTZ AACA------ ---------- ---------- ---------- ---------- ----------neurotactin ----CACAGA CCCCTCTGTG AAGCCACAGA CCCCTCCCAG CTCTCGTGGG GAGCC~AAGG

P1 CTTGGACCCA GAGCTCTCAC CTCCAGCCAT AGCTGCCAGG CACAAGACCC TCAGTGCAAASNTZ ---------- ---------- ---------- ---------- ---------- ----------neurotactin CTTGGACCCA GAGCTCTCAC CTCCAGCCAT AGCTGCCAGG CACAAGACCC TCAGTGCAAA

P1 CGGGATCTGG AGAGAAGAAA CCCTCATGTT CCCGTGGTGG GAAGCAGGAG AGCTACACGGSNTZ ---------- ---------- ---------- ---------- ---------- ----------neurotactin CGGGATCTGG AGAGAAGCAA CCCTCATGTT CCCGTGGTGG GAAGCAGGAG AGCTACACGG

P1 AGAAGGATGA TGGGTGGTCA GTTTCACTCC TGGCTGCCTA CAGCATCCTC CGGAGCAACGSNTZ ---------- ---------- ---------- ---------- ---------- ----------neurotactin AGAAGGATGA TGGGTGGTCA GTTTCACTCC TGGCTGCCTA CAGCATCCTC CGGAGCAACG

P1 CAGCTGCAGC AGCTCATTCC AGGAGTATGA AGCCCAGACA GGACTCCAGC TTCTATGTATSNTZ ---------- ---------- ---------- ---------- ---------- ----------neurotactin CAGCTGCAGC AGCTCATTCC AGGAGTATGA AGCCGAGACA GGACTCCAGC TTCTATGTAT

P1 CTCCACAATC ATGTGACGAC AGGCAGGAGG CGGAGSNTZ ---------- ---------- ---------- -----neurotactin CTCCACAATC ATGTGACGAC AGGCAGGAGG CGGAG

Fig. 3 Pairwise alignment ofthe 3′ insertion of SNTZ forPrecious 1 (P1) with originalviral SNTZ vector, and chickenneurotactin (yellow homologoussequence with predicted neuro-tactin, turquoise homologoussequence with SNTZ)

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from a single cut within the 5′ viral LTR (Fig. 1). South-ern analysis after digestion with EcoRV produced twojunction fragments consistent with a single insertion. Last-ly, the DNA of P1 and P2 were digested with HinDIII toexcise lacZ (approximately 3 kb). Chicken genomic DNAis difficult to completely digest with restriction enzymes,and hence undigested DNA hybridizing with the lacZprobe was present at the top of all lanes. Most impor-tantly, the pattern of restriction fragments for the twofounder birds and their offspring was the same, indicatingthat the DNA was being transferred from parent to off-spring in a stable manner. Finally, the number and size ofthe restriction fragments was the same between P1 and P2suggesting that both animals contained the same site ofproviral integration.

Flanking sequence to the lacZ insert

The region 5′ to the proviral insert was amplified fromP1 transgenic chicken DNA by I-PCR. Portions of theflanking sequence showed a high degree of homology be-tween the partial sequence available for the SNV-basedSNTZ vector (Mikawa et al. 1991, 1992) used to generatethe birds (Mozdziak et al. 2003a), thereby verifying the

presence of the viral LTR. However, BLAST analysis(Altschul et al. 1990) indicated that a region of approxi-mately 180 bp within the vector sequence did not showhomology to the SNTZ vector but instead showed homol-ogy to genomic DNA for Simian Virus (SV) small T anti-gen 40 (SV40). The 180-bp homologous to SV40 wasprobably the nuclear localization sequence in the SNTZvector. The sequence 5′ to the viral LTR showed a highdegree of homology with the predicted neurotactin/frac-talkine in the chicken genome (Fig. 2). Furthermore, thesequence 3′ to the insert also showed a high degree ofhomology to neurotactin/fractalkine (CX3CL1; Fig. 3).The sequenced I-PCR results for P1 and P2 were identical,supporting the results from blot analysis showing that the

Fig. 4 a SNTZ insertion intochromosome 11 (XbaI restric-tion site used for I-PCR, blacklines chicken genomic DNA,lacZ the lacZ gene, LTR viralLTR). The DNA bases of thesequence of the insertion site areshown (underlined DNA bases a5-base repeat of genomic DNA).The 5-bp repeat of genomicDNA is a characteristic sitefor integration of the SNV(Panganiban and Talbot 1993).b lacZ insertion in the genome.The lacZ appears to be insertedbetween exon 1 and exon 2 ofneurotactin on chromosome 11.ENSEMBL numbers are indi-cated for the predicted neuro-tactin and plasmolipin genes

Fig. 5 Representative PCR genotyping gel. A single band in onelane at approximately 500 bp indicates a non-transgenic bird. A lanewith a band at approximately 1,200 bp and a second band at ap-proximately 500 bp indicates a bird hemizygous for lacZ. A lanewith a single band at 1,200 bp indicates a bird homozygous for lacZ

Fig. 6 Neurotactin expression. RT-PCR amplification spanningexon 1 to exon 3 for wild-type (lane 1), hemizygous (lane 2), andhomozygous (lane 3) individuals. RT-PCR amplification spanningexon 2 to exon 3 for wild-type (lane 4), hemizygous (lane 5), andhomozygous (lane 6) individuals. Negative control reactionswithout reverse transcriptase for the exon 1–3 primer set (lane 7)and the exon 2–3 primer set (lane 8). PCR amplification of DNAspanning exon 1 to exon 3 for wild-type (lane 9), hemizygous (lane10), and homozygous (lane 11) individuals. PCR amplification ofDNA spanning exon 2 to exon 3 for wild-type (lane 12), hemi-zygous (lane 13), and homozygous (lane 14) individuals. RT-PCRamplificationofD-glyceraldehyde-3-phosphatedehydrogenase (lanes15-17) with the RNA presented in lanes 1–6 as a template to verifyfidelity

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transgene was probably inserted into the same place in bothfounder animals. The insertion appeared to map betweenexon 1 and exon 2 of the predicted neurotactin/fractalkine(CX3CL1; Ensembl ID no. ENSGALG00000001395) onchromosome 11. The insertion also appeared to be about5 kb from a predicted plasmolipin (Ensembl ID no.ENSGALG00000001391; Fig. 4). The alignments arepresented in Figs. 2, 3 to show homology between theinsertion site and the predicted sequence. However, lacZappears to be inserted in a reverse orientation with respectto neurotactin.

Genotyping

G3 progeny were produced from the P1G2 generation byinter-mating the G2 animals. From a total of 361 G3progeny, 82 were homozyous for lacZ (22.7%), 97 werewild-type non-transgenic (26.9%), and 182 (50.4%) wereheterozygous for lacZ, consistent with the expected Men-delian ratio of 1:2:1 (chi-square: P>0.10). Figure 5 illus-trates a representative genotyping analysis.

Neurotactin expression

Reverse transcription/PCR (RT-PCR) was employed toamplify a 462-bp region corresponding to the predictedsequence spanning exon 1 to exon 3 of neurotactin in thebrain of wild-type chickens. Neurotactin transcripts wereamplified in the brain from hemizygous and wild-typechickens but were not detected in the brain RNA fromhomozygous chickens (Fig. 6). Similarly, RT-PCR wasused to amplify a 309-bp region spanning exon 2 to exon3 of the predicted neurotactin sequence; neurotactin was

only discovered in RNA isolated from the brain of wild-type and hemizygous chickens. PCR spanning exon 1 toexon 3 of DNA revealed the predicted 3.2-kb sequenceonly for wild-type and hemizygous individuals, whereasPCR did not result in an amplification product for thehomozygous individuals, further verifying the insertionsite of the gene. The PCR conditions employed would nothave amplified the predicted fragment (>6 kb). Therefore,we expected that a PCR product would not be producedfrom a homozygous individual. PCR amplification of theregion of DNA spanning exon 2 to exon 3 revealed thepredicted 654-bp sequence for wild-type, homozygous,and hemizygous individuals.

LacZ expression

Beta-galactosidase expression was evaluated from tissuesderived from a 28-week-old P1G3 transgenic chicken andwas detected in all tissues examined in this study. However,expression was qualitatively higher for skeletal muscleand liver of the transgenic birds compared with other tis-sues (Figs. 7, 8). Protein from skeletal muscle was eval-uated from five G2 chickens (>2 years of age) to verifyexpression across individuals (Fig. 9). Beta-galactosidasewas detected in embryonic whole-mounts (Fig. 10). Beta-galactosidase expression was also histochemically detectedin all tissues stained with X-Gal. However, beta-galacto-sidase was not detected in every nucleus of every cell byusing X-Gal (Fig. 11). This was not surprising given thathistochemical staining for beta-galactosidase underesti-mates the number of beta-galactosidase positive nuclei(Couffinhal et al. 1997); an antibody-based detection sys-tem may stain all nuclei. Overall, the range of tissues ex-pressing beta-galactosidase suggests that the birds/embryoswill be a useful model system.

Fig. 7 Western analysis for beta-galactosidase (arrow) from skeletalmuscle (M) and liver (L) from P1G3. Protein samples were analyzedon the same blot presented in Fig. 8 but required a shorter exposuretime to generate an acceptable signal

Fig. 9 Western analysis of beta-galactosidase expression (arrow)from skeletal muscle (lanes 1-5) from five different G2 individuals(lane B control protein from a barred Plymouth rock wild-type non-transgenic chicken, lane C from a non-transgenic wild-type whiteleghorn chicken)

Fig. 10 Whole-mount of chick embryos stained with X-Gal(Control wild-type embryo, lacZ transgenic embryo)

Fig. 8 Western analysis for beta-galactosidase expression (arrow)from heart (H), gizzard (G), small intestine (I), and brain (B) fromP1G3 and from non-transgenic control skeletal muscle (C). Proteinsamples were analyzed on the same blot presented in Fig. 7 butrequired a shorter exposure time to generate an acceptable signal

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Analysis of cell fate

One aspect research at our laboratory centers on is skeletalmuscle biology. Therefore, we undertook a preliminaryexperiment to demonstrate the utility of the transgenic sys-tem for understanding cell fate. We implanted myogenicsatellite cells derived from post-hatch transgenic chickensinto hearts of stage 14 (Hamburger and Hamilton 1951)embryos and detected their presence in day 7 and 14embryos. Whole-mount embryos and PCR analysis for the

transgene revealed that the implanted cells survived in theheart and could be detected by standard methods of anal-ysis (Fig. 12).

Discussion

The development of transgenic chickens has been signif-icantly more challenging than the development of trans-genic mammals. For most mammalian species, foreignDNA is routinely introduced into the genome by micro-injection into newly fertilized zygotes (Gordon et al. 1980;Brinster et al. 1981; Wagner et al. 1981). Although DNAmicroinjection has been a successful approach in birds(Love et al. 1994; Ono et al. 1994), its use has not become aroutine procedure. However, several recent reports havedemonstrated that the production of transgenic chickens byinjecting viral vectors into the embryo of a freshly laid eggis possible (Harvey et al. 2002a,b; Harvey and Ivarie 2003;Mozdziak et al. 2003a; Mozdziak and Petitte 2004; Rappet al. 2003; Kwon et al. 2004; McGrew et al. 2004). Theemphasis of recent transgenic chicken work has been todevelop birds, as proof-of-principle, for the production oftherapeutic proteins. Whereas the domestic chicken is apotential bioreactor for the production of pharmaceuticalproteins, the focus of the current work has been to producea line of transgenic chickens as a useful tool for futurestudies. The present study extends the verification pres-ented in the previous report (Mozdziak et al. 2003a) show-ing that a useful transgenic model has been created to studydevelopment in the chick embryo and in birds after hatch-ing. In addition, the present work characterizes the inser-tion of the transgene, reports PCR genotyping of offspring,and provides further verification of protein expression inadult animals.

Mozdziak et al. (2003a) demonstrated the Mendelianinheritance of the transgene from the G1 to the G2 gener-ation. However, the precise integration site of the SNTZprovirus in the genome was not previously determined.Two different G1 (P1 and P2) roosters containing the lacZgene were produced from a G0 male. The P1 and P2 maleswere the founder animals for all subsequent progeny.Genomic DNA was isolated from each male and digestedwith various restriction enzymes. Southern analysis of P1and P2 and their G2 offspring indicated a single insertionsite that was probably identical between the two birds.Furthermore, the Southern blots clearly showed that thelacZ gene was transferred from the G1 to the G2 gener-ation. The SNTZ flanking sequence further supported theevidence that the lacZ gene was inserted in the same placein P1 and P2 (Figs. 2, 3). Given that the gene was located atthe same location in both P1 and P2, and that a previousstudy had suggested that lactase activity was higher in P1progeny compared with P2 progeny, protein analysis wasrestricted to the P1-derived birds. As the cost of main-taining poultry flocks is high (Fulton and Delany 2003;Miller 2004), only the P1 line will probably be expanded asa homozygous line for future studies. The homozygous P1line is now designated NCSU-Blue1.

Fig. 12 Satellite cells were isolated from skeletal muscle of adultchicken and implanted into stage 14 (Hamburger and Hamilton1951) chick hearts. a Whole-mount of a 7-day embryo stained withX-Gal. b Higher magnification view of heart shown in a Right PCRresults of DNA extracted from hearts implanted with satellite cellsfrom the beta-galactosidase-positive chickens and harvested after 14days of incubation (lane - water control, lane + DNA from thetransgenic chickens, lanes 1–3 hearts implanted with transgenic cellsand yielding negative results, lanes 4–6 hearts implanted withtransgenic cells and revealing the presence of lacZ). All positivelanes contain a 588-bp fragment

Fig. 11 Sections from various tissues of an adult transgenic chickenstained with X-Gal (asterisks seminiferous tubules). Bar 0.1 mm

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Genotyping and proviral integration

For these transgenic birds to be accessible to the develop-mental biology community, identification of the homozy-gous individuals that can form the basis of a breedingprogram to maintain homozygosity is necessary. The eggsof transgenic homozygous birds have been suggested toexpress the transgenic protein at a higher level than those ofheterozygous birds (Harvey et al. 2002a). Homozygousindividuals can be identified through classical test-matingsfollowed by analysis of the progeny. However, classicaltest-mating and subsequent analysis is costly, time con-suming, and laborious, making classical test-matings ofindividual birds impractical. Recently, investigators havesuccessfully employed real-time PCR to identify homo-zygous and heterozygous individuals (Harvey et al. 2002a).Nevertheless, consistent genotyping results have not beenachieved (data not shown) in preliminary studies in ourlaboratory or at a real-time PCR genotyping service lab-oratory at the University of North Carolina Chapel Hill.The inherent challenge to genotyping animals employingreal-time PCR is that there is only a one-copy differencein gene content between homozygous and hemizygousindividuals. In the current study, two different laboratorieshave found that real-time PCR does not supply a suf-ficient level of precision to be able consistently togenotype the transgenic chickens. Therefore, we had toclone the sequence flanking the proviral DNA to developa PCR-based analysis for genotyping. Although I-PCRhas been successfully employed to identify the integrationsites of transgenes in mammals (Kim et al. 2003; Shin etal. 2004), it has not been previously used to evaluate geneintegration or to develop a test for homozygosity intransgenic poultry. The germline transmission rate fromthe G1 to the G2 generation has previously been reportedto be 48%, which is consistent with the expectedMendelian ratio (Mozdziak et al. 2003a). In the presentstudy, hemizygous G2 offspring have been inter-mated,and the genotypic ratios in G3 are approximately 23%lacZ homozygous, 50% lacZ hemizygous, and 27% wild-type non-transgenic, which is consistent with thepredicted 1:2:1 Mendelian ratio (chi-square: P>0.10).Thus, analysis of any future transgenic chickens shouldinclude I-PCR to determine precisely the location of theinserted transgene, and to allow development of a PCR-based test to genotype individuals.

The lacZ gene appears to be inserted in a reverse ori-entation on chromosome 11 within the predicted first in-tron of neurotactin/fractalkine. The proviral insertion maytherefore have disrupted neurotactin function. Neurotactin,also known as fractalkine, is a chemokine that has beenproposed to have roles in neuronal network organizationand neuronal cell adhesion, but its function has not beenfully elucidated in mammals or birds. Although the trans-genic birds appear normal, neurotactin may have beeninactivated because it is non-lethal in knockout mice (Junget al. 2000; Cook et al. 2001). Furthermore, based uponRT-PCR results, the predicted neurotactin RNA is presentin the brain of wild-type and hemizygous individuals, but

mRNA transcripts appear to be missing from the brain ofhomozygous individuals (Fig. 6).

Expression

Previously, expression of nuclear-localized beta-galacto-sidase has been reported in Stage 8 (Hamburger andHamilton 1951) embryos and in cultured chicken myo-blasts (Mozdziak et al. 2003a), clearly illustrating that thetransgenic model will be valuable to embryologists andmuscle biologists. However, an evaluation of beta-galac-tosidase expression in adult animals is also necessary.Tissue panels from adult transgenic animals have revealedthe presence of beta-galactosidase protein in all harvest-ed tissues. Beta-galactosidase expression has also beenevaluated by using X-Gal staining and has been detectedin all tissues studied. However, it is not present in everynucleus of every cell (Fig. 11). This is not surprising be-cause histochemical staining for beta-galactosidase under-estimates the number of beta-galactosidase positive nuclei(Couffinhal et al. 1997); an antibody-based detection sys-tem may stain all nuclei. McGrew et al. (2004) haveevaluated transgenic chickens expressing GFP by usingWestern analysis, but in the present study, beta-galactosi-dase expression seems to have been achieved over a widerrange of tissues. Other investigators have limited theirstudies to examining protein expression in eggs (Harveyet al. 2002b; Harvey and Ivarie 2003) or have only exam-ined GFP expression in entire organs (Kwon et al. 2004).Qualitatively, we have seen the strongest expression intransgenic adult animals in the highly metabolically activeliver and skeletal muscle, with lower levels of expressionin other tissues. Whereas differences in beta-galactosidaseexpression occurs across organs and tissues, it is expressedin every tissue. Furthermore, beta-galactosidase expressionappears to be detectable in embryos through adult animals.

Another group also purport to have generated homozy-gous transgenic chickens expressing GFP (Chapman et al.2005). However, Chapman et al. (2005) have not reporteda chi-square analysis of their G1 to G2 data or a Southernanalysis of their data. Although they have probably pro-duced a stable transgenic line, they fail to show convin-cingly (1) the integration of the gene into the genome, (2)Mendelian inheritance through test mating and chi-squareanalysis, and (3) evidence of homozygosity (Chapmanet al. 2005). The stability of the integration event, Men-delian inheritance, and characterization of the insertionevent are highly important aspects of any avian transgenicsystem, because other investigators have dedicated signif-icant resources attempting to generate avian transgenicsonly to find that the transgene has not been stably inte-grated into their model system (Proudman et al. 2001).Therefore, in developing any transgenic system, the inte-gration of the gene into the genome is an essential pa-rameter for analysis.

Historically, the production of transgenic birds expres-sing a reporter gene has been a highly challenging en-deavor. Birds carrying the lacZ gene have been produced

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by infecting primordial germ cells with a replication-defective SNV vector and by transplanting the transformedprimoridal germ cells into early recipient embryos (Vicket al. 1993). However, Vick et al. (1993) have never dem-onstrated beta-galactosidase expression in the offspring.Similarly, Mizuarai et al. (2001) have produced transgenicchickens carrying the GFP gene, but expression has alsonot been reported in their studies. Transgenic chickenscarrying the lacZ gene have been created by using a similarreplication-defective avian leukosis-based retroviral vectorby Thoraval et al. (1995). Unfortunately, beta-galactosi-dase expression has only been noted in cultures of em-bryonic fibroblasts from G2 progeny, and they have notreported expression in the entire embryo. By using micro-injection of DNA into the germinal disk of newly fertilizedeggs, Love et al. (1994) have described the production ofa transgenic rooster carrying the lacZ gene but, again, donot mention beta-galactosidase expression. The presentwork thus presents a thorough analysis of the birds be-yond germ-line transmission of the gene.

In summary, the stable integration and transmission ofthe nuclear-localized lacZ gene from the G1 through theG3 generation has been demonstrated for the transgenicchickens first reported in 2003 (Mozdziak et al. 2003a).Most importantly, the present study is the first to involvethe use of I-PCR to identify the location of a transgene inchickens with the subsequent development of a test togenotype individuals. With the identification of homozy-gous individuals, birds homozygous for lacZ can now bereliably produced. Furthermore, expression of the lacZgene has been evaluated in the embryo and across a var-iety of tissues in adult animals by Western analysis andhistochemistry. Therefore, the utility of the model systemfor embryonic and adult studies has been reasonably wellcharacterized, and eggs will soon become available to theresearch community. The line of chickens has been ex-panded to G4 homozygous individuals and is designatedNCSU-Blue1. Given the availability of the chicken ge-nome database (Hillier et al. 2004) and the importance ofthe chicken as a model system (Brown et al. 2003) forunderstanding gene function, these birds may be valuablein studies set up to answer questions about cell fate andgene function in the chick embryo. Requests for NCSU-Blue1 eggs can be made at: http://www.cals.ncsu.edu/poultry/biotechnology/.

Acknowledgements We thank Dr. Takashi Mikawa, CornellUniversity, for originally producing the SNTZ vector and makingit available to the research community. We also thank Dr. Mikawa forkindly providing us with the sequence of SNTZ. We are grateful toDr. Douglas Rhodes, University of Arkansas, for providing the Taqemployed in this study and to Becca Barnes for managing the flock.

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