Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF)

The cassette with the β-Casein (bovine) promoter and the hG-CSF gene was obtained by digestion of the vector pCR2.1-hG-GSF, kindly provided by the RDA (Rural Development Agency, South Korea), with the enzyme Not I. The electrophoresis of the products of this digestion produced two DNA fragments (4.8 kb of the G-CSF cassette and 5.5 kb of the pCR2.1 vector, Invitrogen). The 4.8 kb fragment were purified from the gel using the Wizard SV Kit (Promega, Fitch-burg, WI, USA). The G-CSF cassette was cloned into the Not I site of the pCiNeo vector (Promega) by the enzyme T4 DNA Ligase (Promega), following the manufacturer’s recommendations (supplementary Fig. S1). The binding system was transformed into E. coli DH5 α by thermal-shock transformation [8]. Ampicillin-resistant (100 η g/mL) colonies had their plasmid DNA extracted by the salting-out method [9]. The isolated clones were confirmed by digestion with the Eco RI enzyme (Promega) which has the ability to discriminate the pCiNeo with the insert and its correct orientation. The final construct (pCiNeo-G-CSF) was confirmed by DNA sequencing (Helixxa Genomic Services S.A., São Paulo Brazil).

Fibroblasts were isolated from a biopsy obtained from the skin of a four years old female Holstein and cultured in vitro according to protocols already established in the Laboratory of Animal Reproduction of Embrapa Genetic Resources and Bio-technology [10]. The isolated cells were cultured in DMEM medium (Gibco BRL, Billings, MT, USA) enriched with 10% fetal bovine serum (FBS) to the 2nd cell pass and frozen in culture medium containing 10% Dime-thylsulfoxide (Sigma, Marlborough, MA, USA), in 0.25 mL vials and stored in liquid nitrogen. Such stock cells were used throughout this work to provide the fibroblasts for insertion of the genes of interest and thereafter as nucleus donors in the TNSC procedures.

Bovine fibroblasts of primary origin, obtained from LRA from skin biopsies, were cultured in vitro (with D-MEM medium + 10% FBS) at 38°C in humidified incubator. These cells were transfected with the DNA fragment containing the hG-CSF cassette and the Neo gene, removed from the pCiNeo-G-CSF vector with Sac I and Bam HI enzymes (Promega). The 6,800 bp fragment containing the G-CSF cassette and the Neomycin/G418 resistance gene (NeoR) was purified from agarose gel (0.8%) with Wizard-SV Kit (Promega), quantified by spectrophotometry and confirmed by agarose gel staining. On the day prior to transfection, 200,000 to 250,000 fibroblasts were seeded into 24 well plates and allowed to adhere to the bottom so that the next day they reached 60%–80% confluence of the well area. In transfections, 0.25 η g of the pEF-GFP (green fluorescent protein) vector (Addgene, Cambridge, Massachusetts, USA) expressing the GFP was used as a positive control. The DNAs were mixed with 100 μL of serum-free D-MEM (Dulbecco Modified Eagle Medium) and subsequently added with 1 μL of Plus reagent (Invitrogen). The DNA/Plus reagent complexes were incubated at room temperature for 5 min. After incubation, 100 μL of D-MEM (without fetal bovine serum-FBS) containing 1.5 μL of Lipofectamine LTX reagent (Invitrogen) was added to the DNA/Plus complex. The DNA/Plus/TX complex was incubated at room temperature for 20 min. Cells were transfected with the DNA/Plus/LTX complex for 6 hours at 38°C. After transfection the medium was replaced by D-MEM + 10% FBS medium and cultured for more 24 h at 38°C. After this time, the medium was replaced with D-MEM + 10% FBS with the antibiotic G418 (Invitrogen) at 0.8 mg/mL. After 8 to 10 days, all cells lacking the integrated NeoR gene in their genome were killed by the G418. As a negative control, we used non-transfected fibroblasts exposed to G418. At two weeks the screening process was completed and the cells made available to verify the presence of the G-CSF/NeoR cassette in their DNA, as well as to be used as donors of DNA in TNCS. The presence of G-CSF/NeoR in the genome of the transfected cells was investigated by PCR, with specific primers, as described before [10].

The bovine ovaries used were collected at slaughterhouse and the follicles measuring from 3 to 8 mm were aspirated with the aid of a 19G needle connected to a vacuum system. The oocytes collected were classified based on the cumulus cell layer and homogeneity of the cytoplasm, and placed in 200 μL drops by 18 to 22 h in in vitro maturation medium, consisting of 199 medium (Invitrogen), supplemented with 10% FBS (Invitrogen), 10 μg/mL FSH (follicle stimulating hormone), 100 IU/mL penicillin and 50 μg/mL streptomycin, covered with mineral oil and kept in incubator at 39°C, 5% CO₂ and saturated humidity.

The SCNT employed in our study was performed as described in the literature [11], with some modifications described previously by our group [12]. After the maturation period, the oocytes were denuded in 0.2% hyaluronidase and selected according to the expulsion of the first polar corpuscle and cytoplasmic quality. Before enucleation (removal of nuclear DNA) the oocytes were incubated for 20 minutes with 2 μg/mL DNA dye Hoechst 33342 and 3.33 μg/mL cytochalasin D, diluted in maturation medium. The enucleations were performed under a Nikon inverted microscope (Nikon, Tokyo, Japan), with the aid of a TransferMAN NK2 electronic micromanipulator (Eppendorf, Hamburg, Germany). The process involves the removal of a small portion of cytoplasmic material accompanied by the metaphase plate adjacent to the first polar corpuscle, confirmed by visualization under UV light of the DNA stained with Hoechst 33342. During reconstruction, the donor cells of the genomic DNA (transfgenic fibroblasts) were inserted into the perivitelline space. The karyoplast complexes were positioned between two parallel electrodes with fusion medium and received DC pulses of 2.5 kV/cm, generated by the BTX-ECM 200 electrofusion apparatus (Harvard Apparatus, Inc., Holliston, MA, USA). After fusion, the karyoplast complexes were artificially activated using 5 μM Ionomycin prepared in SOFaaci [13] with addition of FBS followed by incubation with 2 mM 6-dimethyl-aminopurine (6-DMAP) prepared in synthetic oviductal fluid (SOF) with addition of FBS.

The reconstructed karyoplast complexes were co-cultured in 9.2 cm² Petri dishes (Corning, Midland, Michigan, USA) over a layer of bovine granulosa cells in 160 μL drops of SOFaaci medium supplemented with 5% FBS, covered with silicone oil, and kept in humidified incubators at 39°C, 5% CO₂ and saturated humidity. The development rate were checked on day 2 for evaluation of cleavage, and day 7 for evaluation of blastocyst rate. Nellore heifers were used as recipients for the SCNT embryos. Prior to the embryo transfer, they were submitted to a fixed-time embryo transfer synchronization protocol, which consists of placement of an intra-vaginal implant containing 1 g of progesterone (day 0 –D0) and an i.m. injection of 2 mg of estradiol benzoate in D5, 150 μg of PGF2_α (prostaglandin alfa) was i.m. injected in D8, the progesterone implant was withdrawn in D9 and 1 mg of estradiol benzoate was injected before blastocyst embryos were transferred. On the day of the transfer the recipients were evaluated for location and classification of the corpus luteum size as class I, II, III (large, medium and small respectively). In those who were able to receive the embryo (class I and II), epidural anesthesia was performed with 3 mL of 2% lidocaine hydrochloride in the sacro-coccix space to relax the pelvic region. For anovulation, an applicator was used by inserting the vial containing the embryo and depositing the embryos in the ipsilateral horn to the corpus luteum. After 30, 60, and 90 days after embryo transfer, transverse rectal ultrasonography (Aloka SSD-500, Overseas Monitor Corporation, Richmond, BC, USA) with a 7.5 MHz linear transducer as used to evaluate pregnancy and follow-up the fetal development. Rectal palpation was also used to assess the texture of the placenta, as well as the number and distribution of the placentomes.

Embryo vitrification was performed as previously described by [14], with minor modifications. A holding medium (HM) was used to handle embryos during vitrification and warming procedures, and was composed of HEPES buffered TCM-199 (Invitrogen^®) supplemented with 20% FBS. For vitrification, the groups of embryos were first washed three times in an equilibrium solution composed of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (ME₂SO) dissolved in HM for a minimum of 9 minutes. Embryos were then transferred to a vitrification solution consisting of 15% EG, 15% ME₂SO, and 0.5 M sucrose in HM, and incubated for 45 to 60 seconds. Next, embryos were placed in a Cryotop^® device (Kitazato Corp., Japan) in sets of 3 to 5 embryos under a stereomicroscope. Subsequently, the Cryotop^® was immediately submerged in liquid nitrogen. The vitrified embryos were stored in the liquid nitrogen container, until an adequate moment for transfer into hormone-synchronized female recipients. At that time, warming was performed by immersing the end of the Cryotop^® for 1 minute in a drop of HM supplemented with 1 M sucrose that had been pre-warmed to 37°C. The embryos were transferred to HM supplemented with 0.5 M sucrose for 3 minutes and then finally transferred to original HM. Subsequently, the warmed embryos were cultured for an additional 4 hours on SOF medium. At the end of this period, the embryos were evaluated for re-expansion and developmental stage progression rates. All embryos presenting no morphologically degenerating signals were considered as having survived. Embryos that changed their developmental stage during the culture period between vitrification and post warming evaluation were transferred.

Bovine skin fibroblasts isolated as described above were cultured in D-MEM + 10% FBS medium (Gibco, BRL) and collected at different times of cell growth. This separation was done according to the confluence state, measured in percentage of surface occupation of the cell-culture bottle, as well as the time elapsed after reaching 100% confluence (100% surface occupation by fibroblasts). After collection, the fibroblasts were fixed in 70% ethanol (Sigma) and stained with 500 μg/mL Propionic Iodide (Sigma) solution as described [15]. The amount of DNA / cell content and cell cycle stage classification analyzed by cytometry in Flow Sight cytometer (Amnis-Merck KGaA).

The genetic identity of the cells and animals used in this work: fibroblast of nucleus-donor animal, receptor heifer and transgenic fetus clone was determined by genetic identity examination performed with 16 microsatellite probes certified by ISAG (International Society for Animal Genetics) for cattle. The genotyping and genetic identity report of the DNA samples was issued by Geneal Diagnósticos LTDA. The confirmation of transgene cassette insertion was done by PCR using the primers: G-CSF Exon 4 Foward (5’-CCCACCTTGGACACACTGCAGCTGG), G-CSF Exon 5 Reverse (5’-TCAGGGCTCAGCAAGGTAGCG) and pCiNeo Reverse (5’-GACAAGCTTACACTTTATGCTTCCGGC). PCR (polymerase chain reaction) conditions: 1.25 mM MgCl₂; 0.4 mM dNTP mix; 10 ρmoles of primers and 1.5 units of Taq Polymerase (Invitrogen). Thermal cycling: 95°C / 1 min; 40× (94°C / 20 sec; 65°C / 20 sec; 72°C / 20 sec); 72°C / 5 min; 12°C / ∞.

The transfection of bovine fibroblasts by lipofection was successful, as can be seen in the positive control expressing the GFP, transfected with the LTX reagent and the pEF-GFP vector (Fig. 1A). Transfection with the DNA fragment containing the G-CSF and NeoR expression cassettes was also successful due to the selection response to the antibiotic G418, which eliminated 100% of the untransfected cells in 14 days (data not shown). The DNA of G418 selected cells, containing the hG-CSF/NeoR expression cassette, was submitted to PCR amplification with primers specific for the inserted cassette, confirming the integration of the cassette into the fibroblast genome (Fig. 2, line 1).

Fig. 1. Transgenic GFP fibroblast and SCNT embryos. Bovine fibroblasts transfected with pEF-GFP vector under UV light (A). SCNT blastocyst (D7) embryos produced from GFP fibroblasts under transmitted illumination (B) and UV light (C). Photos taken in Axiovert (Zeiss, Germany) inverted microscope (A) and SteREO Discovery V8 (Zeiss, Germany) microscope (B and C). GFP, green fluorescent protein; SCNT, somatic cell nuclear transfer.

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Fig. 2. hG-CSF expression cassette PCR amplification. Genomic DNA amplified with primers specific for transgenic hG-CSF expression cassette (A), or for endogenous G-CSF bovine gene (B). (1) Transgenic fibroblast line used as nucleus donor; (2) non-transgenic cow donor of the fibroblast line; (3) recipient heifer; (4) transgenic fetus mammary gland; (5) transgenic fetus skin; (5) transgenic fetus muscle; (7) water; (M) 1 kb Plus DNA ladder (Invitrogen). hG-CSF, human granulocyte stimulation factor; PCR, polymerase chain reaction; CSF, colony stimulating factor.

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To confirm that the cells donor used in our SCNT experiments were correctly synchronized in G0/G1, we collected growing cells (60% confluence) and stationary growth phase cells (100% confluence) at different times (0 h, 60 h, 120 h). The collected cells were fixed and stained with Hoechst 33342 and analyzed by flow cytometry to separate the G0/G1 (2N, diploid genome content), S (DNA synthesis) and G2/M (mitosis, 4N genome content) populations. The results indicated that growth inhibition by contact and consequent synchronization of the G0/G1 cell population was efficiently achieved, even within the first few hours after confluence observation (Fig. 3), indicating that the vast majority of cells used in SCNT were at the correct cell cycle stage advocated for a successful SCNT cloning event.

Fig. 3. DNA content profile of bovine fibroblast populations according to the cell cycle stage (G0/G1, S, and G2/M) at 60% confluence (A); or 100% confluence at different post-confluence times: 0 h (B), 60 h (C), and 120 h (D).

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To test our capacity of producing TG-SCNT embryos, bovine fibroblasts transfected with an enhanced GFP (eGFP) cassette, but not selected with G418, were used as a nucleus donor in SCNT procedure. This pilot test produced blastocysts expressing the eGFP reporter gene or not, depending on the incorporation of this cassette into the genome of nucleus donor cells (Fig. 1B and 1C). In a second SCNT assay, the bovine fibroblasts transfected with hG-CSF/NeoR cassette and selected with G418 were used for the reconstruction of karyoplasts, producing 103 embryos, which were transferred to 79 recipient heifers, generating two pregnancies (Table 1). The fetus of the first pregnancy was detected by ultrasonography 30 days after the TG blastocyst anovulation, but it was no longer detected in the subsequent ultrasonography at the 60^th day of gestation. The second pregnancy developed until the seventh month of gestation, when a spontaneous abortion of the cloned TG fetus occurred. During this gestation, the ultrasonography and rectal palpation examination, conducted monthly, showed an anomalous number and distribution of placentomes, indicating a negative prognosis for this gestation. After the spontaneous abortion, genomic DNA was extracted from the aborted fetus tissues: mammary gland, skin and muscle, as well from blood samples of the fibroblasts of the donor cow and the recipient heifer of the TG-clone fetus. The presence of the hG-CSF/NeoR cassette was detected by PCR only in samples from the TG fetus and TG cell line, as expected (Fig. 2). The genetic identification of the DNA samples was done using 16 single sequence repeat (SSR) probes, and the identity between the nucleus donor animal and the aborted fetus was confirmed (Table 2).