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BOR - Papers in Press, published online ahead of print March 24, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028472
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BIOLOGY OF REPRODUCTION 71, 405–409 (2004)
DOI: 10.1095/biolreprod.104.028472
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Generation of Transgenic Cattle by Lentiviral Gene Transfer into Oocytes1

Andreas Hofmann3, Valeri Zakhartchenko4, Myriam Weppert4, Heidi Sebald3, Hendrik Wenigerkind4, Gottfried Brem5, Eckhard Wolf4, and Alexander Pfeifer2,3

Department of Pharmacy,3 Institute for Pharmacology, Center for Drug Research, Ludwig-Maximilians University, 81377 Munich, Germany Institute of Molecular Animal Breeding/Gene Center,4 Ludwig-Maximilians University, 81377 Munich, Germany Agrobiogen GmbH,5 86567 Hilgertshausen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potential benefits of transgenic cattle range from the production of large quantities of pharmaceutically relevant proteins to agricultural improvement. However, the production of transgenic cattle is presently time-consuming and expensive because of the inefficiency of the classical DNA microinjection technique. Here, we report the use of lentiviruses for the efficient generation of transgenic cattle. Initial attempts to produce transgenic cattle by lentiviral infection of preimplantation embryos were not successful. In contrast, infection of bovine oocytes with lentiviral vectors carrying an enhanced green fluorescent protein (eGFP) expression cassette followed by in vitro fertilization resulted in the birth of transgenic calves. Furthermore, all of the calves generated by infection of oocytes were transgenic, and 100% of these animals expressed eGFP as detected by in vivo imaging and Western blotting. In addition, a transgenic calf was produced by infection of fetal fibroblasts followed by nuclear transfer into enucleated oocytes. Taken together, after adjusting lentiviral transgenesis to cattle, unprecedented high transgenesis and expression rates were achieved.

assisted reproductive technology, embryo, gene regulation, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The introduction of genetic material into the germ line of cattle has a huge potential to achieve agricultural improvements as well as biomedical progress. However, transgenesis in cattle is extremely expensive because of the inefficiencies of the presently available techniques. The most widely used transgenic technology today is DNA microinjection into the pronuclei of zygotes (DNA-MI) [1]. The basic principle of DNA-MI is simple; however, a large number of embryos are lost during the procedure and gene transfer rates are very low. Only ~1% of the injected embryos develop into transgenic animals, and approximately half of these animals express the transgene [2]. Therefore, the production costs for transgenic cattle amount to ~$300 000–500 000 per animal. A promising alternative to mechanical gene transfer is the use of viral vectors to deliver transgenes. Although the use of vectors derived from simple retroviruses, such as Moloney murine leukemia virus (MLV), resulted in a dramatic increase in gene transfer rates, retroviral transgenesis is impractical, because retroviral vectors are silenced during embryonic development [3] or shortly after birth [4]. Here, we demonstrate that use of vectors derived from lentiviruses, which are complex retroviruses (for a review, see [5]), achieves transgene integration and expression in cattle with unprecedented high efficacy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Virus Production

The lentiviral vectors (LV-GFP) used are based on a recently described vector system [6, 7] and carry the enhanced green fluorescent protein (eGFP) reporter transgene, a central DNA flap, and the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) [6, 8]. To achieve ubiquitous transgene expression, the vector construct contains the promoter of the human phosphoglycerate kinase 1 gene [6, 7]. Recombinant lentivirus was produced as previously described [7].

Oocyte Recovery and Lentivirus Injection

Bovine cumulus oocyte complexes (COCs) were collected by aspirating ovarian follicles obtained from slaughtered animals and matured in vitro for 22 h in modified TCM 199 (Invitrogen, Karlsruhe, Germany) at 39°C in 5% CO2. For zygote injection, COCs were first cocultured with frozen-thawed semen (106 spermatozoa/ml; capacitated in a swim-up procedure) for 18 h (in vitro fertilization, IVF), followed by complete removal of the cumulus cells through vortexing and then subzonal injection with glass capillaries containing lentiviral vectors (109 infectious units (IU)/ml; a volume of ~100 pl was injected). For oocyte injection, cumulus cells were completely removed by vortexing in the presence of hyaluronidase (Sigma, St. Louis, MO) before subzonal virus injection and 18 h of IVF. Embryos were cultured in modified synthetic oviduct fluid (SOF) supplemented with 10% (v/v) estrous cow serum (ECS) [9] at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.

Infection of Bovine Fibroblasts

Bovine fibroblasts were seeded in 6-well tissue culture dishes and maintained in 3 ml of Dulbecco modified Eagle medium (DMEM; Sigma) in the presence of 10% fetal calf serum (FCS; Biochrom AG, Berlin, Germany) and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin, Biochrom AG). 30%–40% confluent cells were infected overnight in a volume of 300 µl DMEM/10% FCS with lentiviral vectors (~1 x 107 IU). The number of eGFP-positive cells was quantified by FACScan (Becton Dickinson, Heidelberg, Germany).

Nuclear Transfer

Transfer of fibroblast nuclei into enucleated oocytes was carried out essentially as previously described [10]. In brief, 24 h after maturation (2 h postfusion), the fused karyoplast-cytoplast complexes (KCCs) were activated by a 5-min incubation in 7% ethanol followed by a 5 h incubation in 10 µg/ml cycloheximide and 5 µg/ml cytochalasin B. KCCs were then transferred into 100-µl drops of SOF medium supplemented with 5% (v/v) ECS, covered by paraffin oil, and cultured for 7 days at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Day 7 blastocysts were transferred nonsurgically into synchronized recipient cows.

Embryo Transfer

Cyclic heifers (Deutsches Fleckvieh) between 22 and 36 mo old at stage of diestrus were treated with a single dose of 500 µg Cloprostenol (Estrumate; Essex Tierarznei, München, Germany). Animals were observed for sexual behavior (i.e., toleration, sweating, vaginal mucus) to determine standing heat, which occurred around 60 h after Estrumate injection. Seven days later, Day 7 blastocysts were transferred nonsurgically. The recipients were examined on Day 28 after embryo transfer by ultrasonography for the presence of a conceptus, and by palpation per rectum at Days 42, 60, and 90 of gestation. All experiments were performed according to the relevant guidelines for the care and use of animals. All procedures were in concert with the recommendation of the Tierschutz-Informations-Zentrum für die Biomedizinische Forschung der Medizinischen Fakultät (Animal Welfare Information Centre for Biomedical Research of the Faculty of Medicine) of the Ludwig-Maximilians-Universität in Munich.

In Vivo Fluorescence Imaging

Green fluorescence was observed in live animals using a Schott 2500 light source and a 485-nm filter (Zeiss, Jena, Germany). The emitted fluorescence was visualized using a long-pass filter (HQ 500; Zeiss), and images were taken with a Canon Power Shot G2 Digital Camera (Canon, Krefeld, Germany).

Immunohistochemistry

Tissues were fixed with 4% paraformaldehyde (Sigma), cut on a cryostat at 10 µm, and stained with antibodies against eGFP (Clontech, Palo Alto, CA), followed by incubation with secondary biotinylated antibodies (goat anti-mouse; Dianova, Hamburg, Germany), and staining with ABC solution (Vector, Burlingame, CA) and 3',3-diaminobenzidine (270 µg/ml; Sigma).

Detection of transgene expression in spermatogonia was done by staining with TRITC-conjugated Dolchios biflorus agglutinin (DBA; Sigma) [11]. Expression of eGFP in the DBA-stained sections was visualized by direct fluorescence.

Southern Blotting

BamHI-digested DNA was separated by electrophoresis and transferred to Gene Screen Plus Hybridization Transfer Membranes (PerkinElmer, Boston, MA). The blot was hybridized with a full-length 32P-labeled eGFP cDNA probe.

Western Blotting

Tissue samples were minced in lysis buffer (0.5% Triton X-100, 150 mM NaCl, 2 mM CaCl2, and protease inhibitors). After separation on SDS-PAGE and transfer to PVDF membranes (Immobilon-P; Millipore, Bedford, MA), eGFP was visualized using eGFP antibodies (Clontech) and ECL (Amersham).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The lentiviral vector (LV-GFP) used is a self-inactivating (SIN) vector based on HIV-1 and carries eGFP reporter gene under the control of the phosphoglycerate kinase 1 gene promoter [6]. The SIN mutation in the lentiviral long terminal repeats (LTRs) deletes the viral enhancer and promoter sequences [12, 13] that have been shown to be the major targets for the host gene silencing apparatus [14]. In addition, LV-GFP carries a central DNA flap and WPRE to enhance integration and transgene expression [6, 8].

Lentiviral vectors have been successfully used to generate transgenic rodents [7, 15] and swine [16] by infection of zygotes. Therefore, we initially infected bovine zygotes (n = 357) by injecting the viral particles into the perivitelline space that lies between the zona pellucida and the cell membrane (subzonal injection). 22% of the infected embryos developed into blastocysts. Transfer of 17 blastocysts into 10 heifers resulted in the birth of 4 calves. Southern blotting revealed that none of the calves generated by lentiviral infection of zygotes were transgenic (data not shown).

Next, we infected bovine oocytes with lentiviral vectors, followed by IVF. Metaphase II oocytes lack a nuclear envelope, which might hamper lentiviral integration. A total of 48 oocytes were infected by subzonal injection with LV-GFP, and within 7 days after in vitro fertilization 12 blastocysts developed. Fluorescence microscopy revealed eGFP expression in 10 (83%) of these blastocysts. Eight of the eGFP-positive blastocysts were transferred into four heifers. Overall, three pregnancies were diagnosed by ultrasonography after 21 days of gestation. These three pregnancies were carried to term and four healthy animals were born. Southern blot analysis revealed the presence of multiple proviral integrants in all calves born (Fig. 1A), with an identical proviral integration pattern in all tissues of each calf analyzed (data not shown). In vivo imaging in live animals revealed expression of eGFP in all four calves (Fig. 1B), which was confirmed by Western blot analysis (Fig. 1C). Transgene expression was followed over a 5 mo period by Western blotting of skin biopsies. During this time eGFP expression was stable (Fig. 1D).



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  		FIG. 1. Generation of transgenic calves by lentiviral infection of oocytes. A) Southern blot analyses of the four calves generated by lentiviral infection of oocytes demonstrates the presence of lentiviral integrants in all animals born. B) In vivo fluorescence imaging showing green fluorescence in the skin, mucosa, and eyes of a transgenic calf (left), but not in the age-matched control animal (right). The inset shows the bright field image. C) Western blotting of skin samples reveals expression of the transgene in all transgenic animals. D) Long-term expression of the transgene. Western blot analysis of eGFP expression in animals no. 561 and no. 562 shortly after birth (nb) and at the age of 5 month (5mo)

Further detailed histological analyses were carried out on two transgenic animals (no. 581, male and no. 221, female). Direct fluorescence and immunohistochemical analysis using eGFP-specific antibodies demonstrated transgene expression in skin (Fig. 2, A–C), pancreas (Fig. 2, D–F), and kidney (Fig. 2, G–I), which are derived from different primary germ layers: ectoderm, endoderm, and mesoderm, respectively. Expression of foreign genes in the bovine mammary gland is the basis for production of large quantities of recombinant proteins in the milk. In transgenic calf no. 221, histological analysis revealed strong eGFP expression in ductal epithelial cells of the mammary gland (Fig. 3, A and B). Germ line transmission is another central aspect of transgenic studies in cattle. Therefore, we analyzed eGFP expression in the testis of a newborn transgenic bull derived from subzonal injection of LV-GFP (animal no. 581). Cells that were eGFP positive were detected by immunohistochemistry in the seminiferous tubules (Fig. 3C). To determine whether or not the eGFP-positive cells are germ cells, we used a specific marker for spermatogonia, Dolichos biflorus agglutinin (DBA). DBA has a specific affinity for bovine gonocytes and spermatogonia during the first weeks after birth [11]. Staining of testis sections with fluorescence-labeled DBA followed by direct fluorescence analysis revealed eGFP expression in DBA-positive cells (Fig. 3D and 3E). These data clearly demonstrate transduction of the bovine germ line by lentiviral infection of oocytes.



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  		FIG. 2. Histological analyses of eGFP expression. Analysis of eGFP expression in transgenic skin (A–C), pancreas (D–F), and kidney (G–I). Hematoxylin and eosin stainings (A, D, G), immunohistochemical stainings (B, E, H), and direct fluorescence analysis (C, F, I) of consecutive sections are shown. Bar = 50 µm



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  		FIG. 3. Expression of lentiviral vectors in ductal epithelium of the mammary gland and in the germ line. A and B) eGFP expression in sections of the mammary gland of animal no. 221. C) Immunohistochemical detection of transgene expression in testicular sections using eGFP-specific antibodies. D and E) Detection of transgene expression (green) in spermatogonia of a newborn LV-GFP calf by DBA staining (red). Bars = 50 µm

Another possible route to producing transgenic cattle by lentiviral gene transfer is the infection of donor cells followed by nuclear transfer (NT). Infection of bovine fetal fibroblasts (BFF) with LV-GFP resulted in more than 85% transduction (Fig. 4, A and B). There were 214 nuclear transfer embryos produced [10] by transfer of infected fibroblast nuclei into enucleated bovine oocytes without further selection. After a week in culture, 76 (36%) blastocysts were obtained; 56 of the blastocysts were transferred into 31 synchronized recipients, resulting in five pregnancies. Although four pregnancies were lost, one calf (no. 991) was born naturally. Similar figures were obtained after transfer of uninfected BFF nuclei [10]. Green fluorescence was observed by in vivo imaging in the skin, oral mucosa and eye of animal no. 991 (Fig. 4, C and D). Histological analyses demonstrated eGFP-positive cells in all tissues analyzed, including skin (Fig. 4, E and F), pancreas, kidney, muscle, and brain (data not shown). Southern blotting showed an identical number of viral integrants in all tissues of animal no. 991 analyzed (Fig. 4G).



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  		FIG. 4. Generation of transgenic cattle via nuclear transfer using lentivirally transduced donor cells. A) Representative fluorescence microphotograph of bovine fetal fibroblasts (BFF) 72 h after infection with lentiviral vectors. The inset shows uninfected control cells. B) Fluorescence-activated cell scan analysis of control (cntr) and LV-GFP infected (LV-GFP) BFF 72 h after infection. C and D) In vivo imaging of eGFP expression in a NT-derived calf (no. 991). E and F) Histological analysis of eGFP expression in the skin of animal no. 991. Scale bar, 50 µm. G) Southern blot analysis of LV-GFP integration in different tissues of no. 991. Lane 1, heart; 2, cerebellum; 3, pancreas; 4, muscle; 5, skin


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report for the first time the production of transgenic cattle by lentiviral gene transfer into bovine oocytes. Previous studies [4] that used vectors derived from simple retroviruses demonstrated silencing of transgene expression shortly after birth. In contrast, use of lentiviral vectors resulted in high transgenesis rates as well as long-term expression of the transgene. Our data demonstrate that lentiviral gene transfer into bovine oocytes either directly via subzonal injection or indirectly through transfer of transduced nuclei into enucleated oocytes can be used to generate transgenic cattle. However, lentiviral transgenesis has to be adjusted to the target species: Lentiviral vectors have been successfully used to generate transgenic rodents [7, 15] and pigs [16] by infection of zygotes. In contrast, lentiviral gene transfer into bovine zygotes, as well as monkey preimplantation embryos [17] did not result in transgenic animals. In the case of cattle, this problem could be overcome by infection of oocytes. During meiosis I of oocytes, the nuclear envelope is dissolved in a process known as germinal vesicle breakdown. Previous studies have shown that retroviruses preferentially integrate into membrane-free chromatin [4]. Although lentiviruses can transduce nondividing cells, because the lentiviral preintegration complex is transported actively into the nucleus [6, 18], lentiviral integration is significantly slowed in the presence of a nuclear membrane [19].

Expression of eGFP in bovine embryos derived from oocyte injection was strong enough to allow for unequivocal identification of transgenic blastocysts before embryo transfer. Thus, transgenesis and expression rates of 83% at the blastocyst stage were further increased up to 100% of the calves born. Because transgenesis and expression rates are the principal determinants of production costs, this technique should dramatically reduce production costs of transgenic cattle by at least one order of magnitude to a few thousand dollars.

Although nuclear transfer using lentivirus-infected cells results in a much lower number of transgenic cattle than direct infection of bovine oocytes, this route of lentiviral gene transfer offers the possibility to generate transgenic cattle on a "knockout" background by infecting donor cells that carry null mutations of bovine genes. However, this might also be achieved by using the ability of lentiviral vectors to deliver small interfering RNAs, inducing RNA interference which has already been demonstrated for mice [20, 21]. One of the few limitations for the use of lentiviral vectors is the ~10-kb size of the RNA genome that can be packaged into the viral particle. The essential viral elements present in the lentiviral vector, such as the LTRs, a packaging signal, a central DNA flap, and a WPRE element, are ~1.5 kb in size. Therefore, the transgene and the internal promoter together have to be smaller than 8.5 kb.

An important issue is the potential risk of lentiviral insertional mutagenesis. All retroviruses, including lentiviruses, integrate into the host genome. On one hand, this is the basis for viral transgenesis and the transmission of the transgene via the germ line. On the other, activation of proto-oncogenes by retroviral insertion can be an initial factor for the conversion of a normal cell into a tumor cell. Recently, two patients treated with MLV-based vectors developed leukemia because of insertions of the MLV provirus in close proximity of a proto-oncogene promoter (LMO2) [22]. This has been estimated to occur in one out of 100 000 insertions [23]. However, Wu et al. found important differences between the integration site preferences of HIV-1 and MLV [24]. MLV integrates preferentially in genomic regions surrounding the transcriptional start site, especially promoter sequences. In contrast, lentiviral vectors have no such bias for promoters [24]. Therefore, use of lentiviral vectors reduces the risk of proto-oncogene activation as compared to vectors derived from simple retroviruses.


   ACKNOWLEDGMENTS
 
We thank Tamara Holy for expert technical assistance. Harald Ludwig and Barbara Vogg took part in initial experiments of this study.


   FOOTNOTES
 
1 Supported by grants from the Bayerische Forschungsstiftung (492/02) and the DFG. Back

2 Correspondence: Alexander Pfeifer, Department of Pharmacy, Institute for Pharmacology, Ludwig-Maximilians University, Butenandtstr. 5 (C), 81377 Munich, Germany. FAX: 49 89 2180 77326; alexander.pfeifer{at}cup.uni-muenchen.de Back

Received: 17 February 2004.

First decision: 1 March 2004.

Accepted: 16 March 2004.


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