Production of transgenic goat ( Capra hircus ) with human Granulocyte Colony Stimulating Factor ( hG-CSF ) gene in Brazil

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100μg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors weremated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine αs1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.


INTRODUCTION
Production of valuable proteins of pharmaceutical interest in the milk of transgenic farm animals has become an attractive alternative to microbial and animal cell bioreactors.The generation of transgenic large ruminants (cattle) is, however, very expensive because of the long gestation period, small litter size and high maintenance costs of this ruminant livestock species.Contrarily, dairy goats are less expensive, have shorter generation times and produce multiple offspring.For these reasons, the use of dairy goats as a bioreactor animal is of interest.There have been several reports on the production of transgenic goats (Ebert et al. 1991, Ebert and Schindler 1993, Gootwine et al. 1997) and the feasibility of the large-scale production for the industrial application has been confirmed (Baldassarre et al. 2003, Baldassarre andKaratzas 2004).
The human Granulocyte Colony Stimulating Factor (hG-CSF) is a hematopoietic growth factor that stimulates the proliferation and the differentiation commitment of neutrophil precursor cells, and enhances some of the functional properties of mature neutrophils (Morstyn and Burgess 1988).Following its production as a recombinant human protein (Souza et al. 1986), hG-CSF has been the most widely used hematopoietic growth factor due to its proven efficacy against different forms of neutropenia and chemotherapy induced leucopenia.Furthermore, hG-CSF stimulates the mobilization of progenitor cells for autologous or allogenic transplantation (Anderson et al. 2006, Viret et al. 2006).The hG-CSF has been cited for use in treatment of other human health problems, such as myocardial infarction (Oh et al. 2006) and cerebral ischaemia (Lu and Xiao 2006).In addition, Ko et al. (2000) have developed a transgenic female goat harboring goat-casein promoter/hG-CSF fusion gene by microinjection into fertilized one-cell goat zygotes.
Following a feasibility study in which transgenic mice that secrete high levels of hG-CSF into their milk were produced (Dvoryanchikov et al. 2005) at the Institute of Biophysics Carlos Chagas Filho (Universidade Federal do Rio de Janeiro, Brazil), we initiated a project to produce hG-CSF-transgenic goats in Brazil.Thus, the aim of this study was to examine the overall efficiency of production of goats that are transgenic for the hG-CSF.This study resulted in the production of a transgenic goat containing a goat s1-casein/human G-CSF fusion gene, the first transgenic goat produced in Latin America.

CONSTRUCTION OF G-CSF EXPRESSION VECTOR
The hG-CSF gene was fused to both goat and bovine DNA sequences of s1-casein gene (CSN1S1).The DNA construct was inserted into a plasmid vector, named pGCm3 (Fig. 1).This expression vector was designed based on pGCm1 and pGCm2 previously described and used for production of transgenic mouse (Dvoryanchikov et al. 2005).The pGCm3 DNA insert (6429 bp) has a 5'flanking fragment (3387 bp) originated from goat CSN1S1 gene (Ramunno et al. 2004).This fragment includes a promoter region, the first exon, the first intron and the 12 bp from second exon.In pGCm3 construct, there is the full-length hG-CSF gene (1504 bp) followed by 3'flanking fragment (1538 bp) originated from bovine CSN1S1 gene.The last sequence includes the exon 18, the intron 18, the exon 19 and a 3'-untranslated region (3'UTR).

DNA PREPARATION FOR ZYGOTE MICROINJECTION
The DNA insert was cut out from pGCm3 using SalI digestion.After fractionating the digests in 0.7% agarose gel, a 6431 bp fragment was isolated.The fragment was eluted from agarose gel using Qiagen columns according to recommendations of the manufacturer.For injections, DNA was diluted in 0.01 M Tris-HCl with 0.25 mM EDTA, at pH 7.4.

EXPERIMENTAL ANIMALS AND ETHICS
A total of 23 adult Saanen goats (1-5 years old) were used as embryo donors in this study, while 48 undefined breed goats (2-5 years old) served as recipients for the microinjected embryos.The study was carried out between mid-January and mid-February 2006.Studies were conducted in conformance with guidelines of animal care.This project was approved by the Animal Ethics Committee of the State University of Ceará (CEUA/UECE) as well as the Biosecurity National Technical Committee (CTNBio).

ESTRUS SYNCHRONIZATION AND SUPEROVULATION
The timing of estrus was synchronized in donors and recipients with intravaginal sponges (Progespon, Syntex, Buenos Aires, Argentina) containing 60 mg medroxyprogesterone acetate for 10 days and an injection of 75 µg cloprostenol (Prolise, Arsa, Buenos Aires, Argentina) on the morning of the eighth day.Donors received a total equivalent to 200 mg NIH-FSH-P1 (Folltropin-V, Vetrepharm, Ontario, Canada) given twice daily in decreasing doses over 3 days (50/50, 25/25 and 25/25 mg) starting 48 h prior to sponge removal.Also 48 h prior to the end of progestagen treatment, recipients were injected with 300 IU eCG (Novormon, Syntex, Buenos Aires, Argentina).Thirty-six hours after sponge removal, donors received 100 mg GnRH (Fertagyl, Intervet, Boxmeer, Holanda).Donors were hand bred at 36 and 48 h after sponge removal using Saanen bucks of known fertility.EMBRYO RECOVERY Donors and recipients were deprived of food and water for 24 h prior embryo recovery and transfer.Embryos were surgically recovered 72 h following sponge removal.A low dose (0.1 mg/kg) of xylazine hydrochloride (Rompun, Bayer, São Paulo, Brazil) was intramuscularly injected as a preanesthetic agent.After a peridural injection (7 mg/kg) of lidocaine (Anestésico L, Eurofarma, São Paulo, Brazil) a mid-ventral incision was made and the reproductive tract was exteriorized.Ovaries were observed for fresh ovulation sites to serve as an estimate of the number of embryos expected.The oviduct was then flushed retrogradely with 10-15 ml of sterile phosphate-buffered saline.The flushing medium was collected into sterile Petri dishes and examined under a stereomicroscope (Nikon SMZ-800, Kawasaki, Japan) for the presence of ova/embryos.

EMBRYO MANIPULATION AND MICROINJECTION
The microinjection was carried out using only fertilized one-cell embryos.In order to visualize pronuclei all zygotes were briefly centrifuged at 13,400 rpm for 4-6 min.After centrifugation, if the pronuclei were not visible, they were placed in droplets of M-2 medium supplemented with 10% FCS and covered with mineral oil, and briefly cultured at 37 • C with 5% CO 2 .The zygotes with visible pronuclei were placed in droplets of M-16 medium supplemented with 10% FCS prior to microinjection, which was performed under inverted microscope with DIC optics (Nikon TE2000, Kawasaki, Japan) and a pair of micromanipulators (Narishige, Tokio, Japan).The DNA fragment isolated from a plasmid containing the pGCm3 gene was injected into the one of two pronuclei in volume of 1-2 pl.Noticeable swelling of the nuclei was the criterion for successful microinjection.Microinjected embryos were cultured for 1-2 h at 37 • C with 5% CO 2 to evaluate post injection survival.The surviving zygotes were maintained in culture until embryo transfer.Non-injected zygotes with invisible pronuclei were further cultured to confirm fertilization by cleavage.
EMBRYO TRANSFER AND PREGNANCY DIAGNOSIS Successfully microinjected embryos were transferred into the oviduct of estrus-synchronized recipient goats following laparoscopic exploration in order to confirm the presence of at least one recent ovulation.For embryo transfer, a mid-ventral laparotomy was established and the reproductive tract was exteriorized.A Tomcat catheter containing the embryos was introduced through the fimbria 2-3 cm into the oviduct and the embryos were injected into the lumen.Three to six embryos were transferred per recipient.Pregnancy was detected by ultrasound at 28-35 days following transfer, using a Falco 100 scanner (Piemedical, Maastricht, The Netherlands) equipped with a transrectal 6-8 MHz linear array.
IDENTIFICATION OF TRANSGENIC GOATS DNA was extracted from skin biopsies taken from the ears of 2-week-old kids.After proteinase-K/SDS treatment, a phenol-chloroform extraction was performed following the protocol of Sambrook et al. (1989).The identification of transgenic animals was carried out by PCR amplification using the following pairs of primers: PrA/PrB and PrC/PrD, originating products of 700 bp and 530 bp, respectively (Table I).The reaction control was made with a pair of primers designed to 5'-flanked sequence of goat αs1-casein gene (496 bp PCR product).The PCR was performed using 1 µg of genomic DNA, 1 µM of each primer and 0.5 U of Taq polymerase to a total volume of 25 µL.The amplification was conducted under following conditions: denaturing for 3 minutes at 95 • C, then 35 cycles (30s at 95 • C, 30s at 55 • C and 30s at 72 • C) and the final stage for 5min at 72 • C. The PCR products were analyzed by electrophoresis in a 1.5 or 3% agarose gel and visualized by ethidium bromide staining.

STATISTICAL ANALYSIS
Data were expressed as mean ± SEM.Differences between means were based on t-test.Probability of < 0.05 was considered to be significant statistically.

RESULTS AND DISCUSSION
The response to superovulation and the results of embryo recovery and evaluations are presented in Table II.All donors showed estrus and were responsive to the superovulation treatment (≥ 5 ovulations/female).The superovulation response and the embryo recovery rate from donors found in the present study were superior when compared to those reported in previous studies (Gootwine et al. 1997, Lee et al. 2000, Freitas et al. 2003).
Altogether 379 oocytes/embryos were recovered, of which 75.5% were fertilized and most were at the one-cell stage.These results can be explained by the effect of GnRH injection on the synchronization of ovulation.This way, the donors were hand mated at the good time in order to guarantee a high fertilization rate.In addition, the use of GnRH following sponge removal influenced the stage of development of embryos recovered, a key parameter for the success of a transgenic founder generation program (Baldassarre et al. 2004).
The same authors, working with BELE goats and the FSH-GnRH protocol obtained almost 80% fertilized oocytes of which the most of them were at the pronuclear stage of development.It has been previously suggested that successful integration of foreign DNA is more likely following microinjection of pronuclear-stage embryos than when performed in two-cell stage embryos (reviewed by Baldassarre and Karatzas 2004).
Our results showed the efficiency of oviduct recovery to obtain a high number of microinjectable embryos (79.5%, Table II).However, the surgical nature of this technique limits the number of procedures performed on the same donor before surgical adhesions render the animal unusable for further embryo recoveries.A further refinement of the pronuclear microinjection technology has been reported in using in vitro produced zygotes from oocytes recovered by laparoscopic ovum pick-up (Wang et al. 2002).
In our experiment, as mentioned above, the microinjectable one-cell embryos were a significant proportion of the recovered oocytes/embryos (Fig. 2A,B).However, previous results had showed that the zygote cytoplasm of Korean native strain (Lee et al. 2000) and BELE (Baldassarre et al. 2004) goats contains numerous lipid and opaque inclusions.The same was observed in this study with Saanen goats.This feature makes difficult the pronuclei visualization before microinjection of recombinant DNA.In order to improve the pronuclei visualization, zygotes were briefly centrifuged as described earlier (Lee et al. 1997).The procedure allowed us to visualize the pronuclei in some zygotes (Fig. 2C,D) so that total number of goat zygotes with visible pronuclei was 55% (77 out of 129).In the remaining zygotes we were unable to observe a detailed morphology of the pronuclei and the microinjections were done in an area where the pronuclei were most probably located.In these cases, it was impossible to control the efficiency of injection by observing such as an important change in pronucleus morphology as its swelling (Fig. 2D).

Analysis of the pronucleus formation in zygotes of
Saanen goats revealed that it was impossible to localize the pronuclei at the early stages of zygote development due to its very small size.After a short-time culture the pronuclei became larger and were clearly visible at about one half of cultured zygotes.The pronuclei Fig. 2 -Goat embryos at one-and two-cell stage (A, ×150 and B, ×200) obtained from two donors.A goat zygote after centrifugation with two visible pronuclei (C) and the same zygote during microinjection (D, ×300).Goat embryos at morula stage after in vitro culture for 96 h (E, ×200)."Carlos": a two-week transgenic goat bearing the pGCm3 transgene (F).

TABLE I
Primers used to identification of transgenic goats.

Name
Nucleotide sequence Position c PrA a 5'-AAAGGATAAGGCTAATGAGG-3' 4543 PrB b 5'-CGTTGTACTTTTGTACTGAGC-3' 5247 PrC a 5'-TCTGCAAAAGCAGGCTAAAGC-3' 3188 PrD b 5'-GCCAAGACACTCACCCATCAG-3' 3718 a = forward primer, b = reverse primer, c = relative to pCGm3 construct.were located near the zygote center emerging from the lipid granules, which were shifted to the zygote poles by centrifuging (Fig. 2C).This stage apparently proceeds shortly the moment of the fusion of male and female pronuclei, and therefore corresponds to the late stages of zygote development.After 96 h of culture, non microinjected goat embryos developed normally until morula stage (Fig. 2E).
The hormonal treatment used for estrus synchronization was effective to induce estrus in 87.5% of goat recipients.The estrus began at 26.0 ± 1.6 h after sponge removal and the ovulation rate was 1.3 ± 0.2.These results were similar to other studies performed in goats (Freitas et al. 1997).
Twenty-seven recipients received the microinjected embryos.The pregnancy rate in this report (37.0 %) was comparable to previous reports (Gootwine et al. 1997, Lee et al. 2000).In our study, it was observed that the number of ovulations influenced the number of pregnant goats (Table III).It was probably due to an elevated plasma progesterone concentration, provided by a high number of corpora lutea, during early pregnancy that could improve embryonic survival and growth (Pope et al. 1995).Altogether 10 recipients were pregnant and produced 12 offspring.PCR analysis (Fig. 3) finally identified one transgenic goat in this study.
Efficient production of transgenic goats is one of the key factors in applying this technology to the produc-An Acad Bras Cienc (2007) 79 (4) tion of human pharmaceuticals or other valuable proteins.Our results show that obtaining a reasonable rate of kidding following transfer of microinjected embryos is possible by using the in vivo embryo production and transfer to recipients with a high number of ovulations.
In the present study one transgenic kid was produced (Fig. 2F).This transgenic goat represents 8.3% of the kids born and 0.7% of the embryos microinjected and transferred.At our knowledge, this is the first report of birth of transgenic goat in Latin America.

Fig. 3 -
Fig. 3 -Identification of transgenic goat using PCR analysis with PrA/PrB (A) and PrC/PrD (B) primers.Control amplifications with specific primers designed to 5'-flanking sequence of the goat αs-casein gene (C).C− = negative control, genomic DNA of wild-type goat; 3 to 16 = DNA sample of experimental goats (4 = "Carlos" positive for the pGCm3 transgene); C+ = a positive control (mixture of non-transgenic goat genomic DNA and the recombinant pGCm3 DNA); W = no template; M= a 100 bp ladder.
This work was supported by grants from Ministério de Ciência e Tecnologia/Rede Nordeste de Biotecnologia/ Banco do Nordeste (MCT/Renorbio/BNB), Conselho Nacional de Desenvolvimento Científico e Tecnológico/ Programa de Apoio ao Desenvolvimento Científico e Tecnológico (CNPq/PADCT) and by the Program of the Russian Academy of Sciences "Dynamics of plant, animal and human gene polls" (Russia).V.J.F.Freitas and A.C.C. Carvalho are senior investigators of CNPq (Brazil).