Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

Animal Transgenesis and Cloning: Combined Development and Future Perspectives.

The revolution in animal transgenesis began in 1981 and continues to become more efficient, cheaper, and faster to perform. New genome editing technologies, especially CRISPR-Cas9, are leading to a new era of genetically modified or edited organisms. Some researchers advocate this new era as the time of synthetic biology or re-engineering. Nonetheless, we are witnessing advances in high-throughput sequencing, artificial DNA synthesis, and design of artificial genomes at a fast pace. These advances in symbiosis with animal cloning by somatic cell nuclear transfer (SCNT) allow the development of improved livestock, animal models of human disease, and heterologous production of bioproducts for medical applications. In the context of genetic engineering, SCNT remains a useful technology to generate animals from genetically modified cells. This chapter addresses these fast-developing technologies driving this biotechnological revolution and their association with animal cloning technology.

Effects of Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15 overexpression on the steroidogenic metabolism in bovine granulosa cells in vitro.

Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P4 ) and estradiol (E2 )) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P4 secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non-described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism.

Genetic switches designed for eukaryotic cells and controlled by serine integrases.

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

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

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

Distribution of 5-methylcytosine and 5-hydroxymethylcytosine in bovine fetal tissue of the placenta / Distribuição de 5-metilcitosina e 5-hidroximetilcitosina em placenta fetal bovina

5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are modified cytosines found in mammals that are involved in the regulation of gene expression. The aim of this study was to characterize the global patterns of 5-mC and 5-hmC of the fetal placenta of Nellore cattle as well as blood and sperm as controls. 5-mC and 5-hmC levels were determined using MethylFlash Methylated/Hydroxymethylated DNA Quantification Kit, respectively. Placenta tissues showed lower levels of 5-mC and 5-hmC compared to sperm. The male cotyledon showed higher levels of 5-hmC than the female. For the first time, the levels of 5-mC and 5-hmC in Bos taurus indicus were characterized, which may contribute to our understanding of the mechanisms of epigenetic regulation in the placenta. The presence of 5-hmC in somatic tissues suggest that 5-hmC has its own biological function and it is not only a byproduct from the oxidation of 5-mC. These results may be of interest in ARTs, especially in cloning in the diagnosis/prognosis of aberrant placentation and the viability of pregnancies.(AU)

5-metilcitosina (5-mC) e 5-hidroximetilcitosina (5-hmC) são citosinas modificadas encontradas nos mamíferos que estão envolvidas com a regulação da expressão gênica. O objetivo do presente estudo foi caracterizar os padrões globais de 5-mC e 5-hmC em placenta fetal de animais da raça Nelore, assim como em sangue e espermatozoides, usados como controles. Os níveis de 5-mC e 5-hmC foram determinados usando os kits MethylFlash Methylated/Hydroxymethylated DNA Quantification, respectivamente. Tecidos placentários apresentaram menores níveis de 5-mC e 5-hmC quando comparados com espermatozoides. Cotilédones de machos apresentaram maiores níveis de 5-hmC do que os de fêmeas. Os níveis de 5-mC e 5-hmC em animais Bos taurus indicus foram caracterizados pela primeira vez, o que pode contribuir para o nosso conhecimento sobre a regulação dos mecanimos epigenéticos na placenta. A presença de 5-hmC em tecidos somáticos sugerem que essa base pode ter sua própria função biológica, sendo não somente um sub-produto da oxidação da 5-mC. Esses resultados podem ser de interesse nas Tecnologias de Reprodução Assistida, especialmente na clonagem, no diagnóstico/prognóstico de placentação aberrante e viabilidade da progênie.(AU)

Mucin 2 (MUC2) promoter characterization: an overview.

Transgenic livestock have been studied with a well-known interest in improving quantitative and qualitative traits. In order to direct heterologous gene expression, it is indispensable to identify and characterize a promoter suitable for directing the expression of the gene of interest (GOI) in a tissue-specific way. The gastrointestinal tract is a desirable target for gene expression in several mammalian models. Throughout the surface of the intestinal epithelium, there is an intricate polymer network, formed by gel-forming mucins (especially MUC2 and MUC5AC, of which MUC2 is the major one), which plays a protective role due to the formation of a physical, chemical and immunological barrier between the organism and the environment. The characterization of the gel-forming mucins is difficult because of their large size and repetitive DNA sequences and domains. The main mucin in the small and large intestine, mucin 2 (MUC2), is expressed specifically in goblet cells. MUC2 plays an important role in intestinal homeostasis and its disruption is associated with several diseases and carcinomas. This mucin is also an important marker for elucidating mechanisms that regulate differentiation of the secretory cell lineage. This review presents the state of the art of MUC2 promoter structure and functional characterization.

Maternal SIN3A regulates reprogramming of gene expression during mouse preimplantation development.

The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A.

The role of oocyte-secreted factors GDF9 and BMP15 in follicular development and oogenesis.

Ovarian physiology is controlled by endocrine and paracrine signals, and the transforming growth factor ß (TGFß) superfamily has a pivotal role in this control. The Bone morphogenetic protein 15 (BMP15) and Growth differentiation factor 9 (GDF9) genes are relevant members of the TGFß superfamily that encode proteins secreted by the oocytes into the ovarian follicles. Through a paracrine signalling pathway, these factors induce the follicular somatic cells to undergo mitosis and differentiation during follicular development. These events are controlled by a mutually dependent and coordinated fashion during the formation of the granulosa cell layers. Many studies have contributed to our knowledge concerning the paracrine factors acting within the follicular environment, especially regarding GDF9 and BMP15. We aimed to review the relevant contributions of these two genes to animal reproductive physiology.

Animal transgenesis: state of the art and applications.

There is a constant expectation for fast improvement of livestock production and human health care products. The advent of DNA recombinant technology and the possibility of gene transfer between organisms of distinct species, or even distinct phylogenic kingdoms, has opened a wide range of possibilities. Nowadays we can produce human insulin in bacteria or human coagulation factors in cattle milk. The recent advances in gene transfer, animal cloning, and assisted reproductive techniques have partly fulfilled the expectation in the field of livestock transgenesis. This paper reviews the recent advances and applications of transgenesis in livestock and their derivative products. At first, the state of art and the techniques that enhance the efficiency of livestock transgenesis are presented. The consequent reduction in the cost and time necessary to reach a final product has enabled the multiplication of transgenic prototypes around the world. We also analyze here some emerging applications of livestock transgenesis in the field of pharmacology, meat and dairy industry, xenotransplantation, and human disease modeling. Finally, some bioethical and commercial concerns raised by the transgenesis applications are discussed.

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells.

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos.

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression.

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

Adenosine-rich elements present in the 5'-untranslated region of PABP mRNA can selectively reduce the abundance and translation of CAT mRNAs in vivo.

The poly(A)-binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post-transcriptional level. The binding of PABP to the poly(A)-rich element found in the 5'-untranslated region (5'UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine-rich elements in the 5'UTR of the chloramphenicol acetyl-transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5'UTR poly(A) element. Furthermore, one 5'UTR-tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.