Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector
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- Hiripi, L., Negre, D., Cosset, FL. et al. Transgenic Res (2010) 19: 799. doi:10.1007/s11248-009-9356-y
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Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.
KeywordsLaboratory rabbit Lentiviral transgenesis Simian immunodeficiency virus Mosaic expression Germ-line transmission
The rabbit is a standard laboratory animal in biomedical research and transgenic rabbits are used as animal models for a variety of human diseases both genetic and acquired. The rabbit (Oryctolagus cuniculus) is phylogenetically closer to primates than rodents (Graur et al. 1996) and is large enough to permit non-lethal monitoring of physiological changes. For these reasons, a number of research groups have chosen the transgenic rabbits as animal models for the study of lipoprotein metabolism, atherosclerosis, cardiovascular research and hypertrophic cardiomyopathy (Bosze and Houdebine 2006).
The first transgenic rabbits were obtained two decades ago by pronuclear microinjection (Hammer et al. 1985) and although improvements in the methodology have been reported since than (Besenfelder 1998) it is still an inefficient method: only 1% of injected eggs result transgenic founders. Creation of transgenic rabbits by somatic nuclear transfer would be a viable alternative, however, this method is still in infancy. Other alternative methods suffer from variability: recently dimethylsulfoxide-sperm-mediated gene transfer was adapted to produce human recombinant proteins in the milk of lactating does albeit at low levels (Shen et al. 2006).
Lois et al. (2002) and Pfeifer et al. (2002) opened the door for the use of replication defective lentiviral vectors for transgenic applications. The proof-of-concept study was performed by Lois et al. (2002) in mice and rat showing that the injection of VSV-G pseudotyped lentiviral vectors into the perivitelline space of fertilized oocytes could significantly increase production efficiency. Pfeifer et al. (2002) demonstrated that lentiviral vectors can be used to efficiently manipulate the zona-free embryos to produce founder transgenic mice. Since then human immunodeficiency virus (HIV-1)-derived lentiviral vectors were used to achieve reproducible high transgenesis rates in pigs (Hofmann et al. 2003), while transgenic chicken (McGrew et al. 2004) and transgenic pigs (Whitelaw et al. 2004) were created with an equine infectious anaemia (EIAV) based lentiviral vector. Transgenic cattle were successfully created by lentiviral infection of bovine oocytes but attempts with pre-implantation embryos failed (Hofmann et al. 2004). Rhesus monkey (Wolfgang et al. 2001) was the only species, in which the most commonly used HIV-based lentiviral vector injection did not result transgenic founders, however, recently it was successful in another non-human primate species, the common marmoset (Sasaki et al. 2009). In the last 5 years numerous transgenic laboratory and livestock animals were created by HIV-based lentiviral vectors and various tissue specific, cellular and viral promoters have been examined to direct transgene expression (Park 2007), but the creation of transgenic rabbits has not been reported so far. We aimed to use the lentiviral technology to increase the efficiency of transgenic rabbit production. In vitro data published earlier indicated that contrary to other mammalian cell lines, rabbit cells are poorly permissive to HIV-1 vectors, but could efficiently be infected with simian immunodeficiency virus (SIV) vectors (Hofmann et al. 1999). More recently, Cutino-Moguel and Fassati (2006) described a HIV-1 specific post-entry block in rabbit cells, which resulted aberrant trafficking of HIV-1 and was independent of the envelope construct used, suggesting that lentivirus vectors other than HIV-1 could be more appropriate to transduce rabbit cells. SIV-derived replication defective lentiviral vectors have now been generated in several laboratories. Characterization of these vectors showed that they are similar to HIV-derived vectors with respect to the insertion of transgenes in non-proliferating cells (Negre and Cosset 2002). SIV vectors perform better than HIV-1 vectors in simian cells (Negre et al. 2000) and were successfully used in the transplantation of genetically modified CD34+ cells to reconstitute the myeloid and lymphoid compartments (Derdouch et al. 2008). For creating transgenic animals we used SIV vectors for the first time and report here the establishment and germ-line transmission of the GFP marker gene in rabbits, created with SIV vector based lentiviral transgenesis.
Materials and methods
SIV-derived lentiviral vectors
A second generation packaging system and a monocistronic SIV transfer vector was utilized: the SIV-GAE-CAG-GFP-WPRE-SIN LTR (Negre and Cosset 2002). The CAG is a robust promoter, consisting of the CMV enhancer, the chicken beta-actin promoter and the rabbit beta globin intron. The method for generating SIV-based vectors has previously been described (Negre and Cosset 2002). Briefly, the virus particles pseudotyped with vesicular stomatitis virus G glycoprotein were produced by transient co-transfection of 293T vector producing cells using the calcium-phosphate method (Kvell et al. 2005). Their supernatant was concentrated 1000× fold by gradient-ultracentrifugation over a layer of 20% sucrose and titrated on Jurkat cells by measurement of GFP positive cells by FACS to establish transducing units per ml (TU/ml). The viral titre was found to be 1 × 106 TU/ml; the viral stock was re-suspended in DMEM and stored at −80°C.
Zygote collection and transduction
Collection of rabbit zygotes and transfer of injected embryos to recipient does was performed as described earlier (Bodrogi et al. 2006). Lentiviral vector injection was performed based on the modified perivitelline space injection method to produce transgenic mice from low titre lentiviral vector (Ritchie et al. 2007), injecting ~300–500 pl into the perivitelline space of each single cell embryo. Creation and handling of lentiviral transgenic rabbits was performed under biosafety level 2 precautions. All experiments were approved by the Animal Care and Ethics Committee of the Agricultural Biotechnology Center and complied with the Hungarian Code of Practice for the Care and Use of Animals for Scientific Purposes, including conditions for animal welfare and handling prior to slaughter.
Genotyping, identification of transgenic founders and their progeny, and determination of transgene copy number
Offspring derived from recipient does were screened for transgene integration by transgene specific PCR of genomic DNA purified from ear punch tissue or blood. GFP specific PCR was performed as published (Kvell et al. 2010).
Lentiviral transgene integration number was determined by Southern blot analysis of DNA from tail biopsy. 20 μg of DNA was digested with BamHI, separated on a 1% agarose gel, blotted to nylon membrane and probed with 282 bp-long PCR amplified GFP fragment using primers F: 5′-ctcgtgaccaccctgacctac-3′ and R: 5′-catgatatagacgttgtggctgtt3′.
GFP auto-fluorescence was detected using blue light illumination (GFP excitation frequency 455–495 nm with a barrier filter cut-off below 500 nm. For newborn and adult rabbits and wet tissues this was with a GFSP-5 headset (Biological Laboratory Equipment, Maintenance and Service Ltd., Budapest). Photomicrographs of the embryos were taken with Olympus BH2 research microscope, Zeiss Axio Imager microscope or using the MAA-03/B universal light source (Biological Laboratory Equipment, Maintenance and Service Ltd., Budapest) for Olympus SZH Stereo Zoom microscope.
GFP protein detection with western analysis
50–100 mg rabbit tissue samples were homogenized in eight volumes of ice cold PBS buffer. After centrifugation (12,000g for 1 min) the supernatants obtained from transgenic and non-transgenic animals were separated on 12% denaturing SDS–polyacrylamide gels. The proteins were transferred to PVDF membrane (Hybond-P, Amersham), blocked for 1 h with 5% nonfat dry milk. The blots were incubated for 1 h with a mouse anti-GFP monoclonal primary antibody (JL-8, Clontech, dilution: 1:10,000), washed, and then incubated for 45 min with a peroxidase conjugated sheep anti-mouse IgG secondary antibody (A5906, Sigma, dilution: 1:10,000). The blots were developed using the ECL-Advance chemiluminescence detection system (Amersham) in a dilution of 1:20 and Hyperfilm ECL autoradiography film (Amersham).
Generation of transgenic rabbits
Transgenesis rate and expression of GFP transgene
Each rabbit born from the embryo transfers was analysed for the presence of the transgene by GFP specific PCR. Among the 87 offspring, 28 were found to be transgenic by PCR. GFP expression was evaluated only in the live-born transgenic founders (F0), first macroscopically by in vivo fluorescence imaging. Young, hairless founders showed individual patterns of mosaic green fluorescence as shown on Fig. 1B (a–d), suggesting that transgene integration occurred only at later stages of embryonic development. Eighteen from the 28 PCR positive live-born transgenic animals expressed GFP, indicating that the chromosomal integration site influenced the GFP expression as expected. Some of the adult transgenic animals showed green fluorescence in the areas not covered by hair: ears, nose and eyes (Fig. 4a), while others were not fluorescent macroscopically (data not shown). Southern analysis showed one transgene integration site (Supplementary Fig. 1), which was expected with this low viral titre. The transgenic animals appeared healthy, growth and reproduction rates did not differ from the transgenic rabbits and their non-transgenic littermates.
Variations in macroscopic GFP expression macroscopically and among the organs of the founders
Germ-line transmission of the SIV-CAG-eGFP transgene
Germ-line transmission rate in SIV-CAG-eGFP transgenic founders
In vivo GFP expressing/total born offspring
Transgenica/total born offspring
SIV3 BT male
SIV 9 JBK male
SIV10 JBK female
The present study examined the feasibility of using a SIV-based lentiviral construct to create transgenic rabbits. The first lentiviral gene-transfer vectors were derived from HIV-1 (Naldini et al. 1996). However, it is known that lentiviruses have a restricted species tropism. HIV-1 is potently restricted in rabbit cells at a post-entry stage, although infection by the SIVmac and MuLV vectors remains efficient (Hofmann et al. 1999). Recent data were shown, that the post-entry block is independent of the cell receptor used by the virus for entry, and was characterized by aberrant intracellular trafficking and impaired chromatin integration of HIV-1 (Cutino-Moguel and Fassati 2006). On the other hand, to-date all reports on lentiviral transgenic mammals—except for one with the EIAV virus (Whitelaw et al. 2004)—have used HIV-1 based vectors (for review Park (2007). Out of the five different ubiquitous promoters, which have widely been used in different lentiviral transgenic models, we chose the CAG promoter. The CAG promoter was used to create transgenic rabbits through traditional microinjection and an ubiquitously GFP expressing transgenic line was created (Takahashi et al. 2007). In our lentiviral construct, the SIV-based vector carries a central polypurine track (cPPT) and the post-transcriptional regulatory element of woodchuck hepatitis virus to increase transduction efficiency and transgene expression level (Negre and Cosset 2002).
The high pregnancy rate and large litter size, which we experienced, could be attributed to the reduced physical intervention of perivitelline space injection compared to pronuclear microinjection, which we did not take into consideration in the number of transferred embryos. In this exploratory study we transferred 20–24 embryos per recipient doe, which is more than a doe can normally take to term and nurse, therefore this resulted a high number of stillborns. It is important to note that the high number of stillborns was not due to or correlated with transgene integration/expression. It is possible therefore, that if fewer embryos were transferred per recipient female even greater overall efficiencies could have been achieved.
Although 6% GFP expressing transgenic founder rabbits is considerably higher than the 1% achieved by pronuclear microinjection, it is much lower than the reported figures—up to 97%—reached in transgenic porcine established both with HIV and EIAV lentiviruses (Hofmann et al. 2003; Whitelaw et al. 2004). This relatively low success rate could be the consequence of the low titre of our lentivirus compared to titres between 109 and 1010 TU/ml in the cases mentioned above. Nevertheless the low virus titre did not result, per se mosaic expression pattern. This is not the intrinsic property of the SIV-CAG-GFP vector either, since a transgenic rat founder created with the same batch of this lentivirus, revealed uniform GFP expression and the transgene inheritance followed Mendelian law through three generations (Bender et al. manuscript in preparation).
Western analysis underlined the quantitative variations in GFP protein expression among different founders and within the different organs of the same founder. The differential expression between the different organs was not the result of tissue specific epigenetic silencing, since we could show that the non-expressing tissues did not have integrated transgenic sequences. We assume that it is the consequence of delayed lentiviral integration in later developmental stages, with individual variance in individual rabbit embryos. The differential timing of pre-implantation development could partly explain the rabbit specific mosaic integration: it was published at first, half a century ago and confirmed recently, that rabbit embryos undergo rapid series of cell division and reach 4-cell stage within 11 h, contrary to rodent, sheep, swine and monkey embryos for which it takes 30–40 h following fertilization (Sultana et al. 2009; Witschi 1956). Recent data by Zaitseva et al. (2009) pointed out that while importin 7-depletion impaired HIV-1 function, other lentiviruses like HIV-2, SIV and EIAV were not affected. It was hypothetised that lentiviruses other than HIV-1 may have evolved to use alternative nuclear import receptors. Rapid early rabbit embryonic development combined with yet un-characterized rabbit-specific nuclear import receptors of the reverse transcription complex might result mosaic transgene expression, not reported in other species. We can not rule out the possibility that another non HIV-1 based lentiviral vector could have been more efficient in creating transgenic rabbit than SIV. Nevertheless our data clearly show that the SIV lentiviral vector based transgene is able to integrate and to be expressed in tissues derived from all three primary germ layers, in the male germ cells and in the extra-embryonic tissues of rabbit. Creation of transgenic rabbits and germ-line transmission of the transgene was achieved with an SIV-based lentiviral vector, in which the GFP marker gene was placed under the CAG promoter. In conclusion lentiviral transgenesis was successful in rabbit for the first time.
We thank Ariberto Fassati (University College, London, UK), Bruce Whitelaw and Bill Ritchie (Roslin, UK) and Marielle Afanassieff (INRA, Lyon, France) for the advices and helpful discussions. The authors thank G. Takács for the artwork. Supporting grants: OTKA T049034, GVOP-3.1.1.-2004-05-0071/3.0 and OM-00118/2008, OTKA PD78310.