MOLECULAR REPRODUCTION AND DEVELOPMENT 60:433±438 (2001)

Expression of a Reporter Gene After Microinjectionof Mammalian Artificial Chromosomes IntoPronuclei of Bovine ZygotesBIN WANG,1* A. LAZARIS,1 M. LINDENBAUM,2 S. STEWART,2 D. CO,2 C. PEREZ,2 J. DRAYER,2

AND C.N. KARATZAS1

1Nexia Biotechnologies, Inc., Ste-Anne de Bellevue, Canada2Chromos Molecular Systems Inc., 8081 Lougheed Highway, Burnaby, Canada

ABSTRACT The introduction of mammalianartificial chromosomes (ACs) into zygotes representsan alternative, more predictive technology for theproduction of recombinant proteins in transgenicanimals. The aim of these experiments was to examinethe effects of artificial chromosome microinjectioninto bovine pronuclei on embryo development andreporter gene expression. Bovine oocytes aspiratedfrom 2±5 mm size follicles were matured in vitro for22 hr. Mature oocytes were fertilized in vitro withfrozen- thawed bull spermatozoa. Artificial chromo-some carrying either b-galactosidase (Lac-Z) gene orgreen fluorescence protein (GFP) gene were isolatedby flow cytometry. A single chromosome was micro-injected into one of the two pronuclei of bovine zygotes.Sham injected zygotes served as controls. Injectedzygotes were cultured in G 1.2 medium for 7 days.Hatched blastocysts were cultured on blocked STO cellfeeder layer for attachment and outgrowth of ICM andtrophectoderm cells. The results showed a high zygotesurvival rate following LacZ-ACs microinjection (74%).However, the blastocyst development rate after 7 daysof culture was significantly lower than that of shaminjected zygotes (7.5 vs. 22%). Embryonic cellspositive for Lac-Z gene were detected by PCR in threeof nine outgrowth colonies. In addition, GFP geneexpression was observed in 15 out of 85 (18%)embryos at the arrested 2-cell stage to blastocyststage. Six blastocysts successfully outgrew, threeoutgrowths were GFP positive for up to 3 weeks inculture. We conclude that the methodology for artificialchromosome delivery into bovine zygotes could lead toviable blastocyst development, and reporter geneexpression could be sustained during pre-implantationdevelopment. Mol. Reprod. Dev. 60: 433±438,2001. ß 2001 Wiley-Liss, Inc.

Key Words: arti®cial chromosome; transgenesis;embryo development; cattle

INTRODUCTION

Transgenic animals play a vital role in basicresearch, agriculture, and pharmaceutical industries.

Many discoveries regarding mammalian gene functionshave been made by modifying animal genomes throughtransgenic animal technologies. Furthermore, life-sav-ing drugs and biomaterials are currently under devel-opment using transgenic animals as bioreactors.Following the report of Palmiter et al. (1982) on ratgrowth hormone expression in mice produced by directDNA injection into zygotes, transgenic animals havebeen generated in rabbit, pig, sheep (Hammer et al.,1985), goat (Ebert et al., 1991), cattle (Hill et al., 1992)by similar methods. However, DNA integration intothe genome occurs randomly and segregation of thetransgene into cells is mosaic in most cases. In recentyears, new generation of transgenic technologies hasemerged, including sperm-mediated gene transfer,retrovirus-mediated gene transfer, transfection of em-bryonic stem cells and chimeric formation, animalcloning by nuclear transfer, and arti®cial chromosomesfor gene transfer (Yang et al., 2000). It is anticipatedthat the perfection of these technologies will eventuallyovercome the shortcomings of conventional DNA micro-injection method.

Mammalian arti®cial chromosomes (MACs) repre-sent a promising alternative means of introducinggenetic material into embryos or zygotes for cellularprotein production and genetic therapeutics (Brown,1992; Willard, 1996; Huxley, 1997; Vos, 1998). MACscan replicate alongside the host chromosomes withoutintegrating into endogenous genome and have theability to carry large amount of custom designed DNAwith regulatory domains enabling tissue-speci®c andlong term expression of gene products (Fabb et al.,1997). The satellite DNA-based arti®cial chromosomesare generated by targeting heterologous DNA to aregion near the pericentric heterochromatin of acro-centric chromosomes, thereby inducing DNA ampli®ca-tion, centromere duplication and de novo formation ofnovel MACs. Successful development and large-scale

ß 2001 WILEY-LISS, INC.

*Correspondence to: Bin Wang, Nexia Biotechnologies, Inc., 21,025route transcanadienne, Ste-Anne-de-Bellevue, QC, Canada H9X 3R2.E-mail: bwang@nexiabiotech.com

Received 2 April 2001; Accepted 11 June 2001

puri®cation of satellite DNA-based arti®cial chromo-somes have been reported (Hollo et al., 1996; deJonget al., 1999). Successful generation of transgenic miceand transmission to progeny of the microinjectedarti®cial chromosomes have been reported recently(Co et al., 2000).

In this study, we describe that arti®cial chromosomescarrying reporter genes can be ef®ciently transferredinto bovine embryos by microinjection and can sustainreporter gene expression during early embryonicdevelopment and extended embryo outgrowth culture.

MATERIALS AND METHODS

In Vitro Maturation and Fertilization ofBovine Oocytes

Bovine ovaries were collected from a local abattoirand transported to the lab in D-PBS (Gibco, NY) atabout 358C in a thermos. Oocytes were aspirated fromantral follicles (2±5 mm diameter) within 1±2 hr afterovary arrival. Oocytes with more than three layers ofcumulus cells were selected. The oocyte maturationmedium was TCM199 with Earle's salts, 25 mMHEPES, 10% heat inactivated calf serum (Immunocorp,Montreal Canada), 0.1% gentamycin (Gibco), 5 mg/mlLH (Nobl laboratory), 0.5 mg/ml FSH (Nobl laboratory),1 mg/ml estradiol (Sigma, St. Louis, MD), 22 mg/mlpyruvate(Gibco). The culture environment was 5% CO2

and 95% humidi®ed air at 38.58C. After 22±24 hr ofmaturation, oocytes were coincubated in BECM med-ium (Lim et al., 1994) containing 10 mg/ml heparin(Sigma) with 2�106/ml frozen-thawed bull spermatozoafor 17±19 hr.

Arti®cial Chromosomes

The preparation of arti®cial chromosomes containingthe lacZ reporter gene (LacZ-ACs) has been describedin detail elsewhere (Telenius et al., 1999). Brie¯y, amouse/hamster/human hybrid cell line (mMZc1) con-taining a 60 megabase murine arti®cial chromosomeswas derived from cell line H1D3 (Kereso et al., 1996).Cell lines containing LacZ-ACs were generated bytransferring the LacZ-ACs to CHO cells using themicrocell fusion method. Through single-cell cloningtwo cell lines were generated. The ®nal LacZ-ACwas composed of a centromere, telomeres, blocks ofmurine satellite repeats, two regions of heterologousDNA including 6±10 copies of the reporter gene lacZ(b-galactosidase) under the control of SV40 promoterand an antibiotic resistant marker gene (hygromycinphosphotransferase).

A second prototype chromosome (GFP-AC) wasgenerated by co-transfecting into murine A9 ®broblastsa modi®ed pIRES-GFP vector (Clontech, Palo Alto, CA;the NsiI/SmaI fragment containing IRES was deleted)and a 9-Kb Not I murine rDNA fragment. Theheterogeneous population was enriched for cells expres-sing GFP under the control of CMV promoter throughfour cycles of ¯ow cytometry (FACs vantage, BectonDickinson, NJ) and expansion. Single cell clones were

established and analyzed by ¯uorescent in situ hybridi-zation (Telenius et al., 1999), using probes for mousemajor satellite DNA, pSAT1 (Wong and Rather, 1988)and pIRES-GFP. One cell line carrying a GFP-ACderived from the breakage of a dicentric chromosomeas a result of ampli®cation of pericentric heterochroma-tin (Hollo et al., 1996; Kereso et al., 1996) was isolatedfor further use.

Large Scale Arti®cialChromosome Isolation

LacZ-ACs and GFP-ACs were puri®ed by ¯ow cyto-metry as previously described (deJong et al., 1999).Flow cytometry sorted arti®cial chromosomes werepelleted by centrifugation of approximately 106 chro-mosomes at 2500g for 15 min at 48C in PAB buffer(pH 7.5±7.8) to a ®nal 10±20 ml of `loose' pellet. Thecomposition of PAB buffer was: 15.0 mM Tris-HCl,0.1 mM EDTA, 20.1 mM NaCl, 20.4 mM spermine,50.9 mM spermidine, 1% Hexylene Glycol, 0.1 MGlycine, 0.945 mM EGTA, 0.15 mM KCl, 0.0004%b-ME, 0.0009% TX-100, 9.45 mM MgCl2, 0.19 mg/mlChromomycinA3, 0.0095 mg/ml Hoechst 33258,0.047 mM NaCitrate, and 0.12 mM NaSulphite.

Microinjection

The microinjection procedure was performed asreported previously (Co et al., 2000) with additionalmodi®cations. The borosilicate glass microtubings(Pyrex, Corning No.77, 1.0 mm O.D.�0.75mm I.D.)were pulled and beveled to a micropipette with a 3±5 mm ori®ce, followed by siliconization treatment. Asiliconized depressed slide was used as microinjectionchamber. The micromanipulation drop consisted of100 ml Emcare (Immuno-Chemical products, NewZealand) containing 1% fetal calf serum and wascovered with mineral oil (Sigma). Five microliters ofconcentrated arti®cial chromosomes in PAB bufferwere mixed with 5 ml of 0.25 M sorbitol (Sigma) solutionin order to facilitate settling of the chromosomes to thebottom of the micromanipulation droplet. At 22±24 hrpost insemination, zygotes were centrifuged at 15,600gfor 7 min to stratify the cytoplasm. A single chromo-some was loaded from the tip into the beveledmicropipette and delivered into one of the pronuclei ofbovine zygotes. Sham injected zygotes, in which asimilar volume of injection buffer was delivered, servedas controls.

Embryo Culture and Embryonic CellOutgrowth Culture

The embryo culture medium was G1.2 supplementedwith 8 mg/ml BSA (Gardner and Lane, 1997). Injectedzygotes were incubated at 38.58C in 5% CO2, 7% O2,88% N2 for 7 days. Hatched blastocysts were culturedon a feeder layer of mitomycin C blocked mouse STO®broblast in ESCM [DMEM (Gibco, BRL, NY) mediumsupplemented with 0.1 mM nonessential amino acids,0.1 mM b-mercaptoethanol and 15% heat inactivatedfetal calf serum] at 38.58C in 5% CO2. Following

434 B. WANG ET AL.

attachment and reaching 70±80% con¯uency (about1000 cells), the monolayer of cells was disaggregatedinto smaller aggregates after incubation in 0.25 %trypsin and 0.04% EDTA solution (Gibco, BRL). Half ofthe cells were passaged onto a fresh feeder ®broblastlayer while the other half was used for PCR analysis.All embryonic cell outgrowth cultures were fed every2±3 days by removing one half of the medium andreplacing it with fresh ESCM medium.

Detection of LacZ Gene andGFP Expression

Embryo outgrowths were screened for the presence oflacZ transgene by PCR using primer set A: 50-CTGGCAAGCG GTGAAGTGCC-30 and 50-CGTAATCAGCACCGCATCAGC-30, which ampli®es a 524 bp fragment of thelacZ gene. A second primer set B: 50-AGGAGCAGAGA-GACAAAGGACATCA-30 and 50-TAGTGGTATTGGTG-GTT GGGGATG-30, which ampli®es a 700 bp portion ofan endogenous gene a-lactalbumin, was used as aninternal control to ensure that the extracted DNAcan be ampli®ed by PCR. Using Amersham PharmaciaPscatawass, NJ) Ready-to-Go PCR beads, 10 ml of eachprimer at a concentration of 1 mM was mixed with 200ng of DNA and was ampli®ed by PCR as follows: Step 1,948C for 5 min; step 2, 948C for 1 min; step 3, 608C for 1min; step 4,728C for 1 min; Step 5, repeat step 2±4 for34 cycles; Step 6, 728C for 7 min; step 7, 48C. Theampli®cation products were analysed by agarose gelelectrophoresis.

Expression of GFP in blastocysts and embryo out-growths was detected at days 7 and 21, respectively(day 0�day of insemination). Detection was per-formed using ¯uorescent microscope with an excita-tion ®lter at 450±490 nm and a bandpass ®lter at510±520 nm.

Statistical Analysis

The percentage data from embryo development invarious stages in lacZ-ACs microinjection groups wereanalysed by single factor ANOVA analysis (Micro-soft Excel). Probability of <0.05 was considered to besigni®cant statistically.

RESULTS

Embryo Development FollowingLacZ-AC Microinjection

During 13 sessions, 396 bovine zygotes were micro-injected with a single LacZ-AC while 496 shaminjected zygotes served as control. The initial cleavagerate of the LacZ-AC injected group was signi®cantlylower than that of the control groups (54 vs. 75%,P<0.05). Following culture, signi®cantly fewer LacZ-AC injected zygotes reached the blastocyst stagethan those in the control groups, 7.5 vs. 22%, res-pectively (P<0.05). Out of 19 outgrown embryonic cellcolonies, nine were subjected to PCR analysis. Three ofnine colonies were found to be LacZ gene positive,indicating that LacZ-ACs had been successfully micro-injected and some had replicated alongside theembryonic cells during their outgrowth after 21 days(Table 1).

GFP Expression Following GFP-ACsMicroinjection Into Bovine Zygotes

GFP expression was detectable in various stages ofembryo development from arrested two cells to blas-tocysts after 7 days in culture. The microinjectedembryos exhibited varying degrees of mosaicism interms of both signal intensity and blastomeres beingpositive for GFP. Out of six blastocysts that outgrewsuccessfully, three were GFP positive (Fig. 1) for up to 3weeks in culture (Table 2). There were no GFP positiveembryos detected in the control group.

DISCUSSION

In this study, we demonstrated that a singlearti®cial chromosome could be microinjected success-fully into bovine embryos, and that the injected bovinezygotes could develop to blastocysts after 7 days in vitroculture. Positive outgrowths for transgene were con-®rmed by PCR analysis. High background staininginterfered with attempts to detect lacZ expression.Detection of lacZ gene expression by X-gal staining inbovine blastocysts and embryonic stem-like cells oftengenerates false positive signals following introducingthe LacZ gene into embryonic cells (unpublished

TABLE 1. Embryo Development and Presence of Transgene Following Microinjection of LacZ-ACs Into BovineZygotes

Groups Replicates

Numberof zygotesinjected

Numberof zygotessurvived

(%)

Embryo stages reached after 7 days inculture (%)

Number ofoutgrowths

Transgenesisrate in

outgrowthcultures(1)2±4c 8±16c M BL

InjectionLacZ-ACs

13 396 293 (74)a 215 (54)a 102 (34)a 51 (17)a 30 (7.5)a 19 3/9 (33%)

Control 13 496 471 (94)b 371 (75)b 226 (47)b 156 (33)b 109 (22)b N.A.(2) N.A.

The data within the same column with different superscript differ signi®cantly (P< 0.05). Sham injected zygotes served ascontrol.(1)Transgenesis rate was tested by PCR using lacZ speci®c primers.(2)Not applicable. There was no attempt to outgrow control embryos.

FUNCTIONALITY OF MAMMALIAN ARTIFICIAL CHROMOSOMES 435

observation). Due to lower signal noise and highersensitivity of the GFP expression assay in embryoniccells (Ikawa et al., 1995; Takada et al., 1997),wechose GFP-ACs to investigate exogenous gene expres-sion in embryonic development following microinjec-tion of arti®cial chromosomes into zygotes. The presentstudy con®rmed that a GFP reporter gene under thecontrol of CMV promoter carried on the ACs wasexpressed for at least 3 weeks. These results indicatedthat the arti®cial chromosomes could replicate anddirect the expression of a reporter gene in hostembryos.

Microinjection is a direct and ef®cient means ofdelivering large fragments of DNA including yeastarti®cial chromosomes (Schedl et al., 1992), mamma-lian chromosome (Li et al., 1996), and MACs (Co et al.,2000). The micropipette tip must be large enoughto accommodate the size of chromosomes in order toreduce the mechanical damage on the DNA, induced byshear forces during injection. However, it is verydif®cult to penetrate bovine zygote plasma membranewith a pipette with large ori®ce since the ooplasmamembrane is very elastic (Gagne et al., 1995). Wefound that bovine zygotes at late pronuclear stage(22±24 hr post insemination) were suitable for ACsinjection. At this particular stage, the membrane couldbe penetrated by a single puncture, and the integrityof the visible ooplasm structure following micro-injection was more resistant to ionic environmentdisturbance created by injection buffer. A high initialsurvival rate of 74% (LacZ-ACs) and 83% (GFP-ACs)post microinjection was obtained in this study. Similarobservations have been reported that zygotes micro-injected between 22±26 hr post insemination devel-oped to blastocysts more frequently than the zygotesinjected at 18 hr (Thomas and Seidel, 1993; Peura et al.,1994).

The majority of transgenic animals generated bypronuclear microinjection appear to be mosaic in bothsomatic cells and germ cells for the pattern of DNAintegration (Wall, 1996). This could be caused by lateintegration of transgenes during embryonic preimplan-tation development (Wall and Seidel, 1992). SinceMACs, such as those used in the present study, do notintegrate into the host embryo genome, the foreigngenes should replicate independently and distributeevenly into every embryo blastomere. However, mostof the GFP positive embryos obtained in this studyshowed various extent of mosaicism. A delay in theonset of replication of the unpaired arti®cial chromo-somes during ®rst few rounds of embryo blastomeredivision (Co et al., 2000) or the unequal separation ofthe sister chromatin into daughter blastomeres during®rst mitosis might account for these results. Further-more, we observed a signi®cantly lower embryo devel-opment rate of initial cleavage and blastocyst formationfrom LacZ-ACs injection groups compared to shaminjection groups (Cleavage: 54 vs. 75%; blastocysts: 7.5vs. 22%). Shonn et al. (2000) recently reported that aninappropriate connection between chromosomes and

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436 B. WANG ET AL.

microtubules can activate the spindle checkpoint inyeast, a mechanism which delays the onset of anaphaseand ensure the ®delity of chromosome segregation. Asimilar mechanism might also exist in mammalianembryonic cells. Therefore, the improper replicationand/or separation of introduced arti®cial chromosomesmight disturb the early embryonic cell cycle regulationmediated by microtubule structure, hence halted theembryo development.

In conclusion, we have demonstrated that MACs canbe ef®ciently injected into bovine zygotes. Expressionof GFP con®rmed that the arti®cial chromosomecould replicate and regulate the expression of areporter gene in bovine embryos. Since the transmis-sion of arti®cial chromosome through the germ linehas been recently documented in transgenic mice(Co et al., 2000), broader applicability of the arti®cialchromosome technology in more predictable expres-sion of recombinant protein production in transgenicdomestic animals should be expected in the nearfuture.

ACKNOWLEDGMENTS

The authors thank Jay Petkau, Diane Monteith,Neil MacDonald, Jennifer Danielson, Gary de Jong,Jennifer Asano, Carol Keefer, and the helpful discus-sions of Gyula Hadlaczky.

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TABLE 2. Embryo Development and GFP Expression Following Microinjection of GFP-ACs Into Bovine Zygotes

GroupsRepli-cates

Zygotesinjected

Zygotessurvived

(%)

# GFP positive embryos/# embryos examined at variousstages of development following 7 days in culture (%)

# GFP positiveembryo outgrowths/

total # outgrowthcolonies examined

(%)1 cell 2±4 cell 8±16 cell Morula Blastocyst

GFP-ACs 3 102 85 (83) 0/20 (0) 3/23 (13) 6/25 (24) 3/8 (38) 3/9 (33) 3/6 (50)Control 3 109 109 (100) 0/25 (0) 0/36 (0) 0/19 (0) 0/11 (0) 0/18 (0) 0

Sham injected zygotes served as control.

FUNCTIONALITY OF MAMMALIAN ARTIFICIAL CHROMOSOMES 437

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438 B. WANG ET AL.