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1 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, China
2 Shanghai Transgenic Research Center, 88 Cai-Lun Road, Shanghai 201203, China
3 Shanghai Genon Bio-Engineering Co. Ltd, 88 Cai-Lun Road, Shanghai 201203, China
Correspondence
Guoxiang Cheng
chenggx{at}cngenon.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
There is now considerable evidence that prions, the pathogens of these diseases, consist solely of an infectious, 
-sheet-rich, protease-resistant conformational isoform (PrPSc) of the cellular prion protein (PrPC, a glycosylphosphatidylinositol-anchored membrane glycoprotein of uncertain function) (Prusiner, 1982; Legname et al., 2004). The ‘protein-only’ hypothesis proposes that PrPSc, when introduced into animals, will cause the conversion of PrPC into a likeness of itself and that animals with no PrPC expression should be resistant to experimental scrapie infection, neither developing symptoms of prion diseases nor allowing propagation of the infectivity. This prediction was testified successfully by two independent lines of mice that are devoid of PrPC. Both lines of mice generated by homologous recombination in murine embryonic stem (ES) cells develop and reproduce normally and are resistant to scrapie (Büeler et al., 1992, 1993; Prusiner et al., 1993; Manson et al., 1994).
Due to the success of murine ES cells, gene targeting has been a routine tool for modifying the genome of mice precisely. In spite of considerable efforts, ES cells that can contribute to the germline of any livestock species are still not available, which limits the widespread use of this technology. With the development of cloning techniques, transgenic livestock can be generated by nuclear transfer from transfected fetal fibroblasts cultured in vitro (Schnieke et al., 1997) and the procedures are essentially the same as those required for gene targeting. This provides an alternative approach to circumvent the establishment of ES cells in livestock to modify their genome precisely, because fetal fibroblasts can be used to replace ES cells in gene targeting. Live gene-targeted sheep with a human 
1-antitrypsin gene inserted into the 1 (I) procollagen locus have been produced successfully by gene targeting on ovine fetal fibroblasts (McCreath et al., 2000). One allele of the (1,3)-galactosyltransferase gene in pigs and sheep has also been disrupted by similar procedures (Denning et al., 2001; Dai et al., 2002; Lai et al., 2002).
Scrapie, the prototype of prion diseases, is a naturally occurring disease of sheep and goats and was also the first prion disease to be transmitted to laboratory rodents (Chandler, 1961). Although targeted disruption of the murine prion protein gene does not have gross deleterious effects on mice carrying this mutation, there is no evidence that targeted disruption of this gene in sheep or goats will be analogous. In addition, there is evidence that goats are less susceptible than sheep to the scrapie agent (Billinis et al., 2002). The availability of sheep and goats with reduced or no expression of PrPC will be helpful in understanding the behaviour and adaptation of the TSE infectious agent in these models. Finally, four PRNP+/– cloned sheep were reported by Denning et al. (2001), but none of the animals survived for more than 12 days, leaving the question: what were the reasons for the death of these PRNP+/– sheep – cloning procedures or the genetic modification itself? In this report, we demonstrate the feasibility of producing live, healthy PRNP+/– cloned goats in which one allele of the PRNP gene has been disrupted functionally.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Gene-targeting vector construction.
The promoterless gene-targeting vector GTPrP was constructed by placing the neomycin phosphotransferase gene followed by a polyadenylation signal (neo-pA) directly adjacent to the endogenous initiation codon of the PRNP locus. The 1·9 kb left arm was amplified by PCR from purified genomic DNA of GFF88 fetal fibroblasts by using primers [PrPLF: 5'-GAATCCGCGGTGTCGACTCTCTAGTCCATGATCGTTCCTC-3', with two artificial restriction-enzyme sites (underlined) at its 5' end for molecular cloning (SacII) or vector linearization (SalI), and PrPLR: 5'-TGTTCAATGGCCGATCCCATGATGACTTCTCTGCAAAATAAAG-3', with a 3' tail (23 bp, in bold) within the PRNP locus and a 5' tail complementary to the start of neo coding sequences]. The 1·1 kb neo-pA fragment was amplified by using primers [neoF: 5'-CTTTATTTTGCAGAGAAGTCATCATGGGATCGGCCATTGAACA-3', with a 5' tail (23 bp, in bold) within the PRNP locus and complementary to the left arm, and neoR: 5'-GAATGCGGCCGCAGTACTCCCCAGCTGGTTCTTTCCG-3', with two sites for molecular cloning (NotI) or genomic DNA analysis (ScaI)]. These two fragments were used to prime from each other to give a 3·0 kb product. This product, digested with SacII and NotI, was ligated to a 4·5 kb right arm also amplified from purified genomic DNA of GFF88 fetal fibroblasts by using primers [PrPRF: 5'-ATAAGCGGCCGCGGATCCAGACTATGAGGACCGTTACTATCGTG-3', with two sites for molecular cloning (NotI) or genomic DNA analysis (BamHI), and PrPRR: 5'-CCGCTCGAGGTCGACGTATCATTCACTTCGGCTCTGTAAA-3', with two sites for molecular cloning (XhoI) or vector linearization (SalI)] to complete the GTPrP vector. The GTPrP targeting vector was linearized with SalI before electroporation.
Transfection and selection of the caprine fetal fibroblasts.
After being linearized with SalI, the GTPrP targeting vector was introduced into passage 3 GFF88 fetal fibroblasts by electroporation. About 1·0x107 cells were harvested at 60–70 % confluence, mixed with 20 µg linearized and purified GTPrP vector, transferred into a 0·4 cm cuvette (Bio-Rad) and subjected to a pulse of 220 V, 950 µF, delivered by a Gene Pulser II (Bio-Rad). The transfected cells were plated into 10 cm dishes in GMEM without selection. After 48 h, all cells were trypsinized and reseeded in selective cell-culture medium with 250 µg G418 ml–1 (Gibco-BRL). After 8–10 days selection, healthy and well-separated colonies were isolated with cloning rings and transferred to 96-well cell-culture plates. At subconfluence, a small number of cells was isolated and transferred to 48-well plates for PCR analysis and the remaining cells were expanded by passaging until sufficient cells were obtained for cryopreservation and DNA extraction for Southern blot analysis.
DNA analysis.
Drug-resistant colonies were first screened for targeting events by three different sets of PCR amplification across the 5'-homologous arm or the 3'-homologous arm. The positions of PCR primers are shown in Fig. 1. About 5000 cells in 48 wells were lysed in 40 µl lysis buffer [40 mM Tris/HCl (pH 8·0), 0·9 % Triton X-100, 0·9 % Nonidet P-40, 0·4 mg proteinase K ml–1] at 65 °C for 30 min and then heated to 95 °C for 10 min to inactivate the proteinase K. PCR amplification was performed in a 20 µl reaction volume using the TaKaRa LA system with 2 µl cell lysate as DNA template. The primer sequences were: P1, 5'-CACAGCCAGGCATTCAGAAAC-3'; P2, 5'-CCACCATGATATTCGGCAAG-3'; P3, 5'-CGCCTTCTTGACGAGTTCTTC-3'; P4, 5'-CACGATAGTAACGGTCCTCATAGTC-3'; P5, 5'-GTATGATGCAGGGAAACCAAAG-3'. The thermal-cycling conditions were: 2 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 3·5 min (P1/P2 and P1/P4) or 5 min (P3/P5) at 72 °C; followed by 10 min at 72 °C. PCR-amplification conditions for purified DNA from tissue or blood were the same as those for cell lysate, except that 1 µl purified DNA was used as template.
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): a 1·9 kb fragment corresponding to the 5'-homologous arm of GTPrP or a 1·1 kb fragment corresponding to the neomycin cassette.
Nuclear transfer.
Saanen dairy goats were used as oocyte donors, temporary recipients and final recipients. These animals were purchased from other farms and were maintained in the Shanghai Nanhui Transgenic Experimental Animal Base of Shanghai Transgenic Research Center for several months before they were used in nuclear transfer. All of the animal work was done following a protocol approved by Shanghai Municipal Experimental Animal Committee. The procedures were carried out essentially as described previously (Zou et al., 2002).
Northern and RT-PCR analysis.
Total RNA was isolated from brain tissue of one naturally dead PRNP+/– goat and one wild-type goat at similar age or from wild-type GFF88 cells by using TRIzol reagent (Gibco-BRL) following the manufacturer's instructions. RNA samples (20 µg) were separated on 1·0 % agarose/formaldehyde denaturing gels, transferred to a nylon membrane and hybridized with four different probes: a 2·9 kb ex3 fragment located in the third exon of the PRNP gene and produced by PCR with a forward primer (5'-GACTATGAGGACCGTTACTATCGTG-3') and a reverse primer (5'-CCAATCCCACCATACACACATC-3'), a 1·1 kb neo fragment, a 143 bp ex1+2 probe containing the sequences of the first two exons of the PRNP gene and produced by RT-PCR with the forward primer (5'-TGCCAGTCGCTGACAGCC-3') and reverse primer (5'-GTGATTCAGCTCAAGTTGGATCTG-3'), and a 470 bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene fragment produced by RT-PCR using forward primer (5'-GCAAGTTCCACGGCACAG-3') and reverse primer (5'-CGCCAGTAGAAGCAGGGAT-3').
RT-PCR was performed by using an RT-PCR kit (TaKaRa) following the manufacturer's instructions. First-strand cDNA was synthesized with 2 µg total RNA by using reverse transcriptase in a volume of 20 µl. Subsequent PCR was carried out by using 2 µl cDNA sample as template. The primer sequences were: P6, 5'-CAACCAAGCTGAAGCATCTGTC-3'; P7, 5'-AAGAACTCGTCAAGAAGGCGATAGAAGGCG-3'. P2 and P4 have the same sequences as were used in the DNA analysis.
Western blot analysis.
For brain tissue, 10 % (w/v) homogenates were made in lysis buffer [10 mM Tris/HCl (pH 7·4), 100 mM NaCl, 10 mM EDTA, 0·5 % Nonidet P-40, 0·5 % sodium deoxycholate] at 4 °C and were centrifuged at 1000 g for 5 min. Then, the supernatant was used for Western blot. Equal amounts of protein were separated by SDS-PAGE (12 % gels) and transferred to PVDF membranes by semi-dry electroblotting (Bio-Rad). After preincubation for 1 h in blocking buffer [50 mM Tris/HCl (pH 7·5), 150 mM NaCl, 10 % non-fat dry milk], the membranes were incubated overnight at 4 °C in the same buffer containing a 1 : 4000 dilution of a mouse anti-prion protein monoclonal antibody (mAb) (4C6, National BSE Reference Laboratory, Tsingtao, China) or a 1 : 1000 dilution of a mouse anti-actin mAb (Sigma, catalogue no. A4700). After washing, the membranes were incubated for 2 h in the blocking buffer containing a 1 : 1000 dilution of the peroxidase-conjugated goat anti-mouse IgG antibody. Then, the membranes were visualized by using SuperSignal West Pico chemiluminescent substrate (Pierce).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Although the whole caprine PRNP gene sequence has not been reported, the ovine PRNP gene has previously been cloned and well characterized. It has three exons spanning 21 kb genomic DNA, with the 770 bp coding region contained entirely within the final exon (Lee et al., 1998). Two homologous arms, a 1·9 kb fragment and a 4·5 kb fragment, of the GTPrP targeting vector (Fig. 1) were generated by PCR from purified DNA of GFF88 cells by using primers that were designed according to reported sequences of the ovine PRNP gene (GenBank accession no. U67922
[GenBank]
). In the constructed GTPrP vector, a 1·1 kb neo-pA cassette was inserted directly adjacent to and in frame with the endogenous initiation codon of the PRNP gene. If homologous recombination occurs between the endogenous PRNP locus and the GTPrP vector, a 436 bp coding region following the initiation codon will be deleted and replaced by the 1·1 kb neo-pA cassette.
Disruption of the PRNP gene with the GTPrP vector
Linearized GTPrP was transfected into early passage fetal fibroblasts by electroporation. A total of four rounds of independent transfection, G418 selection and colony isolation were carried out in three different cell lines (Table 1). We first transfected linearized GTPrP into female GFF88 cells, from which two homologous arms were generated. After 8–10 days drug selection, 163 colonies were isolated. G418-resistant colonies were initially screened by three independent PCRs to detect targeted events. Of 112 colonies analysed by PCR using a forward primer, P1, that is located 5' of the left homologous arm and a reverse primer, P4, that is located within the right homologous arm, ten were found to have undergone the desired recombination event as determined by the presence of two bands of the expected sizes: a 2·8 kb band from the normal PRNP locus and a 3·5 kb band from the targeted PRNP locus. When homologous recombination occurred between the endogenous PRNP locus and the GTPrP vector, about 0·4 kb coding region of the PRNP locus was replaced by the 1·1 kb neo-pA cassette, resulting in a new 3·5 kb band when analysed by PCR using primers P1 and P4. Fig. 2(a) shows the results of several representative colonies analysed by PCR using primers P1 and P4. However, eight of the ten P1/P4 PCR-positive colonies were found to be mixed colonies, as determined by the lower intensity of the 3·5 kb band compared with that of the 2·8 kb band.
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). In addition, the PCR products amplified with primers P1 and P4 were transferred to a nylon membrane and hybridized with a 1·9 kb 5'-arm probe (Fig. 2d), which demonstrated that the 3·5 kb bands generated with primers P1 and P4 were not non-specific bands. Finally, the 3·0 kb PCR products generated with P1/P2 and the 5·3 kb PCR products generated with P3/P5 from GTPrP74 and GTPrP78 were sequenced and the results were consistent with gene targeting at the PRNP locus (data not shown).
Due to the possibility of false positives produced by PCR, Southern blot analysis of the PCR-positive colonies was also employed to confirm successful targeting. The GTPrP78 colony could not be expanded to yield enough cells for genomic DNA extraction for Southern blot. As a result, only the GTPrP74 colony was analysed by Southern blot analysis. Genomic DNA of wild-type GFF88 cells or of GTPrP74 was digested with three different sets of restriction enzymes and hybridized with two different probes (Fig. 3). If the PRNP gene had been targeted successfully, two BglI sites within the 0·4 kb coding region would be deleted and result in a new 12·0 kb BglI fragment from the targeted locus, in addition to the 6·1 kb endogenous BglI fragment from the non-targeted locus, when the digested DNA samples were hybridized with a 1·9 kb 5'-arm probe (Figs 1 and 3a). Similarly, when genomic DNA of GTPrP74 was digested with XbaI or ScaI/BamHI, there was a new 7·4 kb XbaI fragment in addition to the 6·7 kb endogenous XbaI fragment, or a new 5·5 kb ScaI/BamHI fragment in addition to the 4·4 kb endogenous ScaI/BamHI fragment, respectively (Fig. 3a). When the same membrane was hybridized with a 1·1 kb neo probe, only the newly generated fragments of the expected size from the targeted locus were visible (Fig. 3b), which was consistent with successful targeting at this locus and also indicated that only a single copy of the targeting vector had been integrated into the caprine genome.
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). Although a total of six colonies isolated from transfected B38A cells were found to be positive by PCR analysis, all of them died before sufficient cells could be recovered for nuclear transfer or DNA extraction for Southern blot analysis. In addition, 450 colonies were isolated from transfected 1126B cells and 261 colonies were analysed by PCR, but no colonies were found to be positive.
Production of PRNP+/– goats by nuclear transfer
The healthy and targeted GTPrP74 colony was used as karyoplast donor for reconstructing embryos with enucleated oocytes. Two independent groups of oocyte-collection and embryo-transfer procedures were carried out within an interval of about 1 month (Table 2). At the first group of nuclear transfers, a total of 66 morulae or blastocysts were transferred to 30 final recipients, which produced 10 pregnancies at day 35. Two pregnancies subsequently aborted, but no tissue of fetuses could be recovered for DNA analysis. Five pregnancies were maintained to term, resulting in five live births and one stillbirth. Three kids perished soon after birth and the remaining two kids remained alive and healthy for up to at least 4 months. Five live kids were delivered in the second group of nuclear transfers. Two of them also died within 48 h of birth and the other three kids also remained healthy for up to at least 3 months. The major characteristics of the 11 cloned goats were summarized in Table 3.
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; Denning et al., 2001). In addition, no gross abnormalities in the brains of these dead kids, such as vacuolation, neuronal apoptosis or astrogliosis, were evident as judged by microscopic examination of haematoxylin/eosin-stained sections (data not shown).
We performed PCR and Southern blot to analyse these cloned goats. PCR analysis showed that five of the six kids delivered in the first group of nuclear transfer and all five kids delivered in the second group of nuclear transfer had one targeted and one normal PRNP allele (Table 3). Fig. 4(a) shows only the PCR results of the first seven delivered kids. Surprisingly, one non-targeted kid (16A) was also generated, which indicated that the GTPrP74 colony still contained some non-targeted cells in spite of being scored as a non-mixed colony by the P1/P4 PCR analysis. Southern blot results (Fig. 4b) also revealed fragments that were consistent with the presence of one targeted and one normal PRNP allele in three live kids (8A, 14A and 45B).
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; Table 3), as judged by their size and body weight (data not shown). We also did not see any abnormal behaviour, such as ataxia or dementia, in these PRNP+/– goats.
Expression analysis of the PRNP+/– goats
To further confirm that the replacement of 0·4 kb coding region of the PRNP gene by the 1·1 kb neo-pA cassette had disrupted the expression of this gene functionally, we carried out Northern blot, RT-PCR and Western blot analysis of the brain tissue of the PRNP+/– 46A goat, which was delivered after only 131 days pregnancy by the final recipient, PrP46, in the second group of nuclear transfer and died at the second day after birth (Table 3).
Northern blot analysis is shown in Fig. 5(b). Hybridization with the 2·9 kb ex3 probe containing the exon 3 sequences of the PRNP gene detected a 4·2 kb mRNA band in the brain tissue of both the wild-type goat and the 46A goat and in the GFF88 fetal fibroblast cells, consistent with the expression of a normal PRNP allele. When the RNA samples were hybridized with a 143 bp ex1+2 probe containing the exon 1 and exon 2 sequences of the PRNP gene, the sample of the wild-type goat only showed the 4·2 kb band, but the sample of the 46A goat showed another new 1·2 kb mRNA band, which was consistent with a PrP–neo fused mRNA in the targeted PRNP allele (Fig. 5a, b). However, the 1·2 kb fused mRNA from the targeted allele was apparently less abundant than the 4·2 kb endogenous mRNA from the normal allele (Fig. 5b). Whether this reflects different mRNA stability or transcription activity has yet to be determined. Hybridization of the same samples with a 1·1 kb neo probe detected a 1·2 kb mRNA band only in the sample of the 46A goat, again consistent with a PrP–neo fused mRNA.
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). A forward primer (P6) located in the second exon and three reverse primers (P2, P7 and P4) located in the neo-pA cassette or in the third exon were used. Amplification with P6/P4 primers revealed a 536 bp band in both samples, which indicated the expression of the normal PRNP allele. As expected, amplification with P6/P2 or P6/P7 revealed a 670 bp band or an 865 bp band only in the sample of the 46A goat, which further confirmed the presence of a PrP–neo fused mRNA in the targeted PRNP allele.
In order to confirm functional disruption of the PRNP gene at the protein-expression level, Western blot analysis of the brain tissue of the 46A goat was carried out to detect PrPC expression in the brain. Brain tissue isolated from the same region of the wild-type goat and the 46A goat was homogenized and an anti-prion protein mAb was used for detection of PrPC. A typical profile of PrPC with an apparent molecular mass of approximately 28–36 kDa was detected in the brain tissue of both the wild-type goat and the 46A goat (Fig. 5d), but the PrPC expression level in the brain of the 46A goat was apparently reduced, which suggested that the expression of the targeted PRNP allele had been disrupted.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Scrapie, a naturally occurring disease of sheep and goats, is used as the prototype of prion diseases, so sheep or goats with reduced or no PrPC expression will be useful in research on prion diseases.
Due to the low efficiency of homologous recombination in somatic cells, it is essential to use a powerful selection strategy to enrich targeted events. Two fundamentally different enrichment methods have been developed: positive–negative selection (PNS) and promoterless selection. Generally, PNS vectors can achieve enrichments of only two- to fivefold, but promoterless vectors can achieve enrichments of 100–1000-fold (Sedivy & Dutriaux, 1999). Such powerful selection of promoterless vectors, whilst not necessary in murine ES cells because of the intrinsically high recombination efficiency, seems essential for efficient gene targeting in somatic cells. Indeed, of the 23 disruptions made in human somatic dells (Sedivy et al., 1999) and of the three genes targeted in livestock (McCreath et al., 2000; Denning et al., 2001; Dai et al., 2002; Lai et al., 2002), all involved a promoterless strategy. Although a milestone was achieved by Kuroiwa et al. (2004), who succeeded in disrupting one transcriptionally silent gene and one transcriptionally active gene in one bovine cell line by sequential targeting with PNS vectors, we chose to use a promoterless vector in our targeting experiment to ensure successful disruption of the caprine PRNP gene.
The splice-acceptor site of the third exon of the PRNP gene was not deleted in these PRNP+/– goats, because there was considerable evidence that deleting the splice-acceptor site of exon 3 of the murine prion protein gene (Prnp) could cause severe ataxia and Purkinje cell loss in later life of Prnp–/– mice (Sakaguchi et al., 1996). This abnormal phenotype was due to the ectopic expression of another gene (Prnd), which shows some sequence similarity to the Prnp gene and is located 16 kb downstream of the Prnp gene. Deleting the splice-acceptor site of the third exon of the murine Prnp gene causes abnormal exon skipping and formation of chimeric transcripts that place Prnd transcription under the control of the Prnp promoter, resulting in abnormal expression of the Prnd gene in the brain of Prnp–/– mice (Moore et al., 1999; Rossi et al., 2001). Disruption of the Prnd gene can prevent the appearance of this abnormal phenotype in Prnp–/– mice (Genoud et al., 2004). Our RNA analysis shows that the RNA of the targeted caprine PRNP allele is transcribed and cleaved normally (Fig. 5), excluding the possible ectopic expression of the caprine Prnd gene in the brain of these PRNP+/– or prospective PRNP–/– goats. In addition, in contrast to the prion protein gene targeting in mice reported by Büeler et al. (1992), who reported that RNA transcription of the targeted murine Prnp allele was not terminated at the neo polyadenylation site, but instead terminated at the downstream endogenous Prnp polyadenylation site, the RNA transcription of the targeted caprine PRNP allele reported here was terminated effectively at the neo polyadenylation site, resulting in a 1·2 kb, rather than a 4·9 kb, PrP–neo fused mRNA.
One minor defect in our DNA analysis was that no external probe outside the homologous arms was used in the Southern blot analysis of the GTPrP74 colony or the cloned goats. We have tried several external probes outside the 5'-homologous arm, but in all of the lanes, including the wild-type samples and the targeted samples, no specific band could be seen (data not shown). One possible explanation for this is the presence of many DNA sequence repeats in this region (Lee et al., 1998). Although external probes play some roles in excluding possible false positives resulting from random integration, we believe that the data shown in this report support the demonstration of successful gene targeting.
Regardless of the fact that six (one non-targeted and five targeted) of the 11 cloned goats perished after birth, we did not see any abnormal development or behaviour in the remaining five live PRNP+/– cloned goats up to at least 3 months of age (Fig. 4c; Table 3). This was consistent with our expectations, because only one allele at the PRNP locus was disrupted. Indeed, mice with both alleles of the Prnp gene disrupted showed no gross abnormalities (Büeler et al., 1992; Manson et al., 1994); however, in contrast, none of the PRNP+/– cloned sheep reported by Denning et al. (2001) survived for longer than 2 weeks after birth, because of various abnormalities. Our results suggest that the incidence of mortality reported here or by Denning et al. (2001) was not a consequence of the disrupted PRNP gene per se, but was due to the nuclear-transfer procedures and/or the prolonged culture or drug selection of the cells used in nuclear transfer. We think that the following aspects need to be considered carefully to produce viable animals by nuclear transfer. First, the cell line to be used in nuclear transfer needs to be selected carefully. Fetal fibroblasts isolated from fetuses of different genetic backgrounds or isolated at a different time have differing abilities of cell division. Second, the concentration of G418 used should be optimized to kill non-transfected cells in about 7 days. Increased concentrations will kill other cells over a shorter time and result in transfected cells at a low density, which will cause the cells to senesce too quickly. Third, the reconstructed embryos should be cultured in vivo to develop to morulae or blastocysts before they are transferred to final recipients.
We have shown the feasibility of modifying the genome of goats precisely by gene targeting of fetal fibroblasts, and the PRNP gene is also the first gene of goats that has been targeted successfully. We have also presented strong evidence at the RNA and protein-expression levels that demonstrates the functional disruption of one allele of the caprine PRNP gene. These heterozygous PRNP+/– goats are now ready to be used in producing homozygous PRNP–/– goats in which no PrPC should be expressed.
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ACKNOWLEDGEMENTS |
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Received 27 July 2005;
accepted 14 December 2005.
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