Irina A. Serova

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 100 microg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated 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 alphas1-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.

Dynamics of Recombinant hG-CSF in Transgenic Goat: Preliminary Study in the Founder during Hormonally Induced Lactation

This study aimed to characterize the dynamic of human granulocyte colony-stimulating factor (hG-CSF) during artificial lactation in a transgenic founder goat and to assess its potential ectopic expression and health. The female secreted 93.9 to 1,474.6 µg hG-CSF per mL of milk. Two peaks of serum hG-CSF (3,470 and 7,390 pg/mL) were detected in the first half of the lactation. Outside of the lactation, hG-CSF was absent from serum, indicating no ectopic expression. During the treatment to induce lactation, transgenic female presented increased neutrophil and lymphocyte blood counts when compared to nontransgenic female. Despite transient neutrophilia, serum biochemistry profiles indicated normal liver and renal functions. Thus, transgenic goat expressed hG-CSF in quantities sufficient for a commercial bioreactor and remained clinically healthy.

Pronuclear embryo yield in Canindé and Saanen goats for DNA microinjection.

The objective of this study was to examine the effect of donor breed on pronuclear-stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen-cloprostenol treatment. Forty-eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH-FSH-P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte‐colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra‐assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild‐type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin‐based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue‐specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

The Use of Reproductive Technologies to Produce Transgenic Goats

Recombinant DNA technology has revolutionized the production of therapeutic proteins. Thus, genes of a great number of human proteins have already been identified and cloned. The use of farm animals as bioreactors may be the better choice to produce re‐ combinant therapeutic proteins. For this activity, the term “pharming” was created, refer‐ ring to the use of genetic engineering to obtain a transgenic or genetically modified animal. Considering the advantages and disadvantages of livestock species, goats appear as a very good model. In addition, the first human commercially approved biological drug (antithrombin (AT)) was produced from the milk of transgenic goats. The aim of this chapter is to present various reproductive technologies used to obtain transgenic goats secreting recombinant proteins in milk. Initially, this chapter presents the methods for embryo production (in vivo and in vitro) to realize the DNA microinjection in pronu‐ clear embryos. Thus, the techniques of superovulation of donors (in vivo embryo produc‐ tion) and ovarian stimulation for oocyte recovery (in vitro embryo production) are described. Also, the methods for DNA microinjection and embryo transfer are detailed in this chapter. Finally, this chapter describes the reproductive procedures used for obtain‐ ing transgenic goats by cloning.

The comparison of two embryo donor breeds for the generation of transgenic goats by DNA pronuclear microinjection

The aim of the present study was to compare two breeds as embryo donors to produce transgenic goats for the production of human granulocyte colony-stimulating factor. Ten Caninde and 11 Saanen goats were used as donors and receivedahormonaltreatmentforoestrussynchronisation.Thesuperovulationwasinducedwithatotaladministrationof4.4 mg/kgbodyweightNIH-FSH-P1,givenindecreasingdosesover3days.Donorsalsoreceived100 mgofGnRHandtheywere hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after progestagenremovalandthepronuclearembryosweremicroinjected.Fifty-tworecipientsofundefinedbreedwereprepared by receiving the same oestrus synchronisation treatment; however, only 32 were used due to the availability of embryos. Embryosweresurgicallytransferredintotheoviduct.Asignificant(P <0.05)differencewasobservedinthetotalnumberof ovulations when Caninde (12.6 � 6.9) and Saanen (22.5 � 10.0) donors were compared. Concerning the microinjectable embryos, Caninde goats produced a greater number when compared with Saanen females (P < 0.05). Twenty recipients received61Canindeembryosand,ofthose,12kidded,whereasjust12recipientsreceived30Saanenembryosbutjustthree kidded. In total, three transgenic goats were obtained, of which two were healthy Caninde and one stillborn Saanen. It was possible to develop an efficient protocol to obtain transgenic goats for Caninde but not for Saanen breed, for which some variables such as superovulatory regime and time of breeding should be further studied.

Unexpected phenotypic effects of a transgene integration causing a knockout of the endogenous Contactin-5 gene in mice

Contactins (Cntn1-6) are a family of neuronal membrane proteins expressed in the brain. They are required for establishing cell-to-cell contacts between neurons and for the growth and maturation of the axons. In humans, structural genomic variations in the Contactin genes are implicated in neurodevelopmental disorders. In addition, population genetic studies associate Contactins loci with obesity and hypertension. Cntn5 knockout mice were first described in 2003, but showed no gross physiological or behavioral abnormalities (just minor auditory defects). We report a novel Cntn5 knockout mouse line generated by a random transgene integration as an outcome of pronuclear microinjection. Investigation of the transgene integration site revealed that the 6Kbp transgene construct coding for the human granulocyte–macrophage colony-stimulating factor (hGMCSF) replaced 170 Kbp of the Cntn5 gene, including four exons. Reverse transcription PCR analysis of the Cntn5 transcripts in the wild-type and transgenic mouse lines showed that splicing of the transgene leads to a set of chimeric hGMCSF-Cntn5 transcript variants, none of which encode functional Cntn5 protein due to introduction of stop codons. Although Cntn5 knockout animals displayed no abnormalities in behavior, we noted that they were leaner, with less body mass and fat percentage than wild-type animals. Their cardiovascular parameters (heart rate, blood pressure and blood flow speed) were elevated compared to controls. These findings link Cntn5 deficiency to obesity and hypertension.

Morphophysiological effects of insertional mutagenesis of the contactin 5 (Cntn5) gene in transgenic mice

The transgenesis technologies are actively used in different fields of biological studies. The most actively used method to obtain transgenic animals by DNA injection in the zygote pronucleus can be accompanied by the violation of the genes’ function instead of integrating the transgenic construction. In this work, the morphophysiological effects of integrating the transgenic construction providing the production of the human granulocyte–macrophage colony-stimulating factor in the milk of the mouse line (GM9) (the insertion of the construction in the intron of the contactin 5 (Cntn5) gene) were described. It was demonstrated that the insertion of the construction did not result in the Cntn5 knockout. However, the gene transcription in the heart and kidneys of transgenic animals changes compared with wild-type animals, and the spectrum of transcripts also changes, indicating a violation in the Cntn5 gene splicing regulation. It is known from the published data that the Cntn5 polymorphisms are associated with an addiction to obesity and a predisposition to arterial hypertension. We studied the main parameters of the fat metabolism and cardiovascular activity in mice homozygous for the transgene insertion in the Cntn5 gene, heterozygous animals, and wild-type animals. The phenotyping carried out demonstrates that homozygous mice have a smaller body weight than wild-type individuals. And the weight difference between genotypes is determined more by significantly lower fat accumulation than by the lag of mutants in the development of the skeleton and muscles constituting the lean mass. Statistically significant differences in the parameters characterizing the intensity of the blood circulation were found in the studied lines. Homozygous mice exceed wild-type individuals by their arterial pressure values, heart rate, and blood flow rate in the tail vessels. It should be noted that heterozygous individuals have intermediate values between mutants and wild-type according to all the measured morphofunctional parameters.

Mosaic expression of LacZ reporter gene controlled by 5′-regulatory sequences of alpha-S1-Casein gene in transgenic mice

The phenomenon of mosaic expression at the cellular level is frequently observed in tissues and organs of transgenic animals. The report concerns mosaicism in the progeny of five transgenic mouse founders carrying the LacZ reporter gene under the control of 5′-regulatory sequences of bovine and goat alpha-S1-casein genes of various sizes. Cells positive for β-galactosidase E. coli activity were detected in lactating mammary glands of all transgenic females; however, the distribution of positive cells within the mammary glands was variable. We observed two types of mosaics, i.e., lobular (clonal) variegation when most or all lobular cells were positive for β-galactosidase and stochastic mosaicism when only single β-galactosidase positive cells were scattered within mammary glands. It is suggested that the stochastic type of mosaicism is realized in cells at the terminal stage of the differentiation of lactating glands, whereas the lobular one is developed from proliferating precursors capable of forming a whole lobule. The ectopic expression of the reporter gene was detected in the mandibular salivary gland in the offspring of two of the five founders No16 and No37, as well as in ovary follicles at the atrezia stage in the progeny of one of these founders. The low level of ectopic expression means that the 5′-flanked regulatory sequences of alpha-S1-casein gene of various lengths used in the constructs ensure the reliable tissue-specific expression of the reporter gene.

Mosaic expression of LacZ

The phenomenon of mosaic expression at the cellular level is frequently observed in tissues and organs of transgenic animals. The report concerns mosaicism in the progeny of five transgenic mouse founders carrying the LacZ reporter gene under the control of 5′-regulatory sequences of bovine and goat alpha-S1-casein genes of various sizes. Cells positive for β-galactosidase E. coli activity were detected in lactating mammary glands of all transgenic females; however, the distribution of positive cells within the mammary glands was variable. We observed two types of mosaics, i.e., lobular (clonal) variegation when most or all lobular cells were positive for β-galactosidase and stochastic mosaicism when only single β-galactosidase positive cells were scattered within mammary glands. It is suggested that the stochastic type of mosaicism is realized in cells at the terminal stage of the differentiation of lactating glands, whereas the lobular one is developed from proliferating precursors capable of forming a whole lobule. The ectopic expression of the reporter gene was detected in the mandibular salivary gland in the offspring of two of the five founders No16 and No37, as well as in ovary follicles at the atrezia stage in the progeny of one of these founders. The low level of ectopic expression means that the 5′-flanked regulatory sequences of alpha-S1-casein gene of various lengths used in the constructs ensure the reliable tissue-specific expression of the reporter gene.