Fig 3 - uploaded by Ignacio Anegon
Content may be subject to copyright.
Fetal growth of embryos produced by lentiviral transgenesis (TG), in vitro fertilization (IVF) and intrauterine insemination (IUI) measured by ultrasonography. No statistical differences were found among groups for any of the variables
Source publication
Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cel...
Context in source publication
Context 1
... development rate on day 5 was 39.3 % (46/ 117) for the TG group and 39.6 % (44/111) for IVF group (P = NS). During pregnancy, no differences were found for crown-rump length on day 30, while on day 45 TG group showed an intermediate value (50.9 ± 4.9 mm; P = NS) in comparison with IVF group (44.9 ± 3.4 mm) and IUI group (55.1 ± 5.1 mm). Heart beats frequency during the examina- tion period did not differ significantly among groups, ranging from 178.9 ± 21.0 to 192.9 ± 4.4 beats per min for TG group, 176.6 ± 7.2 to 194.4 ± 10.4 for IVF group, and 185.4 ± 4.0 to 192.3 ± 6.3 for IUI group (P = NS). Average diameter of placentomes for TG, IVF or IUI groups was 28.4 ± 3.1, 29.2 ± 2.7 and 30.5 ± 3.1 mm on day 60; 37.8 ± 4.5, 38.1 ± 2.7 and 36.1 ± 2.9 mm on day 75; 38.2 ± 2.4, 37.9 ± 3.7 and 33.3 ± 2.4 mm on day 90; and 34.8 ± 0.7, 32.3 ± 3.0 and 32.5 ± 2.3 mm on day 105, respectively (P = NS). The rest of the measures of fetuses for the three experimental groups did not show significant differences and are shown in Fig. ...
Similar publications
Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of p...
ELife digest
Transgenesis – the ability to insert foreign genetic material (known as transgenes) in to the genome of an organism – has revolutionized biological research. This approach has made it possible for scientists to study the role of specific genes and to produce animal models which mimic aspects of human diseases.
For transgenes to be main...
Silkworm transgenesis scientists have done some genetic modification work on multivoltine silkworms, but that type of silkworms is less commercial feasible. They are easy to manipulate, because they breed all year round. But the commercial silkworm variety must undergo hydrochloric acid treatment at a high temperature to be artificially hatched. Hy...
Genetic modification of living organisms has been a prosperous activity for research and development of agricultural, industrial and biomedical applications. Three decades have passed since the first genetically modified products, obtained by transgenesis, become available to the market. The regulatory frameworks across the world have not been able...
Selectable markers, such as antibiotic resistance or fluorescent protein genes, are widely adopted in transgenesis and genome editing for selecting engineered cells with desired genotype but are limited in choices. We demonstrate here the concept of split selectable markers each allowing for selection of multiple "unlinked" transgenes with one sele...
Citations
... Firstly, we checked the efficiency of their transduction with lentivirus containing GFP reporter in the presence of 10 and 50% of virus supernatant. These types of viral vectors are widely used for gene delivery to mammalian [32,33] and avian [34][35][36] cells. To produce transgenic ...
... Firstly, we checked the efficiency of their transduction with lentivirus containing GFP reporter in the presence of 10 and 50% of virus supernatant. These types of viral vectors are widely used for gene delivery to mammalian [32,33] and avian [34][35][36] cells. To produce transgenic birds, most of these studies injected the virus vectors into newly laid eggs. ...
The last decade was marked by a steep rise in avian studies at genomic and cellular levels. Cell lines are important tools for in vitro studies in cell biology and cytogenetics. We developed a simple method of primary somatic cell culture establishment from the ovaries of the great tits (Parus major) and testes of ten Passerine species, characterized the cellular composition of the ovary-derived lines using RT-PCR and immunolocalization of the tissue-specific markers and tested the efficiency of two methods of genetic transformation of the ovary-derived cell line. We found that the ovary-derived cell cultures of the great tit were composed of fibroblasts mainly, but also contained interstitial and granulosa cells. They were cultivated until the 10th passage without any noticeable decrease in their proliferative activity. The testis-derived cell cultures had lower proliferative potential. However, both ovary- and testis-derived cell cultures provided enough material for high quality mitotic metaphase chromosome preparations. The efficiency of its transduction with lentivirus containing a GFP reporter was very low, while electroporation with episomal vectors expressing GFP resulted in a high yield of GFP-positive cells. The proposed method could be used for the generation of high quality material for various cytogenetic and genomic studies.
... BMPR-1B gene was widely expressed in some tissues such as ovary, testes, ear, hypothalamus and blood, mostly in ovary tissue, which regulated the release of gonadotropin hormones (FSH and LH) [7] . Based on the breed and biological properties in ruminant, we hypothesized that BMPR-1B was a major candidate gene for genetically improving reproductive performance in sheep [8] . Tibetan sheep was one of the original sheep breeds in China, and known as excellent adaptability in Tibetan plateau, however, Tibetan ewes were a low reproduction rate breed, which twin rate was only 3~5 percent [9] . ...
Bone Morphogenetic Protein Receptor-1B (BMPR-1B) is considered as the primary gene in sheep for follicular development and litter size trait. It
has been defi ned as the most major candidate gene for genetic markers of sheep reproductive performance. In our study, polymorphisms in the
BMPR-1B gene 3"-UTR region were investigated in 363 Tibetan sheep (119 Plateau-type Tibetan sheep, 141 Valley-type Tibetan sheep, and 103
Oula-type Tibetan sheep) by DNA sequencing analysis. Two single nucleotide polymorphisms (SNPs) were identifi ed, which were G1339A and
A1354G. The frequencies of SNPs were in Hardy-Weinberg equilibrium (Chi-square test, P>0.05). AA and AG genotypes were found in the A1354G
variant of the 3"UTR region, that AA and A were the preponderant genotype and allele, respectively. The χ2 independence test analyses indicated
that the A1354G variant of BMPR-1B gene 3"UTR region polymorphisms was signifi cantly correlated with litter size in all-types Tibetan sheep (P
... Les vecteurs lentiviraux sont des rétrovirus complexes utilisés pour l'insertion de gènes exogènes et dont le génome viral est réparti en plusieurs fragments pour éviter la formation de virus réplicatifs compétents (Park, 2007). Cette technique a permis de produire des souris (Lois et al. 2002), des vaches (Hofmann et al. 2004) et des moutons transgéniques (Crispo et al. 2015a). La transgénèse à l'aide de transposons d'ADN utilise leur capacité de "couper-coller", qui conduit à l'insertion d'un transgène dans le génome de l'hôte en une seule étape. ...
... Cette plateforme se place à l'avant-garde scientifique au niveau national et régional. Depuis 2007, après avoir produit les premiers animaux transgéniques en Uruguay, nous avons produit différents modèles par micro-injection d'ADN dans le pronucléus ( Figure 1) (Crispo et al. 2013), par ciblage génique dans des cellules souches embryonnaires murines et par utilisation des lentivirus (Crispo et al. 2015a). En 2014, des techniques d'édition de gènes impliquant le système CRISPR/Cas ont été implantées à l'UATE alors que les premières souris génétiquement modifiées par cette technique venaient tout juste d'être rapportées dans le monde (Wang et al.. 2013). ...
... L'efficacité de transgénèse à la fois chez les embryons (97,4 %) et chez les animaux nés (100 %) était très élevée, confirmant ce qui avait été rapporté pour cette technique dans d'autres espèces (Park, 2007). En dehors de la fluorescence, les animaux transgéniques étaient phénotypiquement identiques à leurs homologues non transgéniques (Crispo et al. 2015a). Des cellules de ces moutons ont été transplantées dans des moutons non-transgéniques pour suivre la persistance de la fluorescence. ...
La création d'animaux génétiquement modifiés a permis de réaliser de grandes avancées scientifiques dans des domaines variés. En 2006 nous avons mis en place à l'Institut Pasteur de Montevideo une Unité Technologique capable de conduire un programme de recherche propre et de fournir aux chercheurs d’Uruguay et de la région des modèles de souris génétiquement modifiés. En 2014, nous avons mis en œuvre le système CRISPR/Cas9, ce qui nous a permis d'élargir le cadre de notre activité et de développer d'autres projets impliquant la souris et le mouton, nous positionnant ainsi comme plateforme technologique de référence en Amérique du Sud. La législation relative aux Organismes Génétiquement Modifiés (OGMs) dans plusieurs pays d’Amérique du Sud ne prend en compte que les modifications comportant l'addition d'ADN exogène et en exclut donc l’édition génomique. Cette situation procure un avantage régional favorisant le développement rapide de modèles génétiquement éditées y compris dans des espèces animales de rentes. Mots-Clés : CRISPR, édition génomique, rongeurs, mouton.
... RhoD indirectly affects ewe ovarian folliculogenesis and litter size by participating in the MAPK signaling pathway and in various cellular processes. HSP10 either directly or indirectly participates in cell proliferation apoptosis, inflammatory immune response, and reproductive processes [21,22]. ...
Background:
BMPR-1B is part of the transforming growth factor β super family and plays a pivotal role in ewe litter size. Functional loss of exon-8 mutations in the BMPR-1B gene (namely the FecB gene) can increase both the ewe ovulation rate and litter size.
Results:
This study constructed a eukaryotic expression system, prepared a monoclonal antibody, and characterized BMPR-1B/FecB protein-protein interactions (PPIs). Using Co-immunoprecipitation coupled to mass spectrometry (Co-IP/MS), 23 proteins were identified that specifically interact with FecB in ovary extracts of ewes. Bioinformatics analysis of selected PPIs demonstrated that FecB associated with several other BMPs, primarily via signal transduction in the ovary. FecB and its associated interaction proteins enriched the reproduction process via BMP2 and BMP4 pathways. Signal transduction was identified via Smads proteins and TGF-beta signaling pathway by analyzing the biological processes and pathways. Moreover, other target proteins (GDF5, GDF9, RhoD, and HSP 10) that interact with FecB and that are related to ovulation and litter size in ewes were identified.
Conclusions:
In summary, this research identified a novel pathway and insight to explore the PPi network of BMPR-1B.
... To date, Escherichia coli, yeast, cultured cells, Caenorhabditis elegans, Drosophila, butterfly Junonia orithya, zebrafish, medaka Oryzias latipes, mice, rats, rabbits, lambs, pigs and monkeys are available as GFP transgenic models. 25,[40][41][42][43][44][45][46][47][48][49][50] The laboratory mouse is one of the premier mammalian model organisms, and EGFP is popularly used in producing transgenic or targeted mice to create mutations at base pair resolution and tag different cellular populations. 6 Use of GFP and its color variants as reporters and indicators is challenging genome engineering technology and enabling novel approaches in life science studies. ...
Application of green fluorescent protein (GFP) in a variety of biosystems as a unique bioindicator or biomarker has revolutionized biological research and made groundbreaking achievements, while increasing evidence has shown alterations in biological properties and physiological functions of the cells and animals overexpressing transgenic GFP. In this work, response to total body irradiation (TBI) was comparatively studied in GFP transgenic C57BL/6-Tg (CAG-EGFP) mice and C57BL/6 N wild type mice. It was demonstrated that GFP transgenic mice were more sensitive to radiation-induced bone marrow death, and no adaptive response could be induced. In the nucleated bone marrow cells of GFP transgenic mice exposed to a middle dose, there was a significant increase in both the percentage of cells expressing pro-apoptotic gene Bax and apoptotic cell death. While in wild type cells, lower expression of pro-apoptotic gene Bax and higher expression of anti-apoptotic gene Bcl-2, and significant lower induction of apoptosis were observed compared to GFP transgenic cells. Results suggest that presence of GFP could alter response to TBI at whole body, cellular and molecular levels in mice. These findings indicate that there could be a major influence on the interpretation of the results obtained in GFP transgenic mice.
... Estos vectores se obtienen reemplazando los genes virales gag, pulg y env por uno o más genes, mientras que, el conjunto de genes reguladores necesarios para la encapsulación (secuencia ψ), la transcripción reversa (PBS, R, PPT) y la expresión de los genes (LTR) se conservan [9,11]. Así mismo, el transgén más utilizado, debido a que muestra una expresión estable en células de mamíferos y a que se puede rastrear in situ de forma cuantitativa o cualitativa y no invasiva [12] es de la proteína fluorescente verde (eGFP) derivada de la medusa Aequorea Victoria [13]. ...
... Después de 48 h el medio de cultivo fue centrifugado por 25000 rpm durante 1,5 horas a 4ºC. El pellet de lentivirus se resuspendió en 100μL de DMEM sin SFB y congelado a -80ºC hasta su utilización [12,23]. ...
... En otras especies, las tasas de formación de blastocistos son 76% en embriones de macacos [23] y en cerdos se reporta una tasa 25% [27]. Se ha argumentado que, esta menor tasa de producción de blastocistos en bovinos, sea posiblemente debido a que el genoma lentiviral no es capaz de penetrar de forma eficiente en el pronúcleo bovino [12]. ...
The objective of this research was to compare the efficiency of microinjection of a lentiviral vector carrying the eGFP gene in mature oocytes (PRE) and zygotes (POS) six hours after in vitro fertilization (IVF). For this, 1777 mature oocytes were distributed in 7 treatments, one control (T1), three PRE (T2PRE, T3PRE and T4PRE) and three POS (T2POS, T3POS and T4POS) which were microinjected before (PRE) and after (POS) of IVF. The T4PRE and T4POS treatments were microinjected with the lentiviral vector. The blastocyst production rate at day eight and the expression of the eGFP protein in T4PRE and T4POS were evaluated. No differences were found in the blastocyst production rate between the T1 treatment (20,2%), the culture controls (T2PRE and T2POS) and the microinjection controls (T3PRE and T3POS). The presence of the lentiviral vector decreased (p<0,05) the blastocyst formation rate in the T4PRE and T4POS treatments (5,5 and 3,5%, respectively). All blastocysts produced in T4PRE and 71,4% produced in T4POS expressed the eGFP protein (p<0,05), mainly in the trophectoderm and in the internal cell mass of the blastocyst.
... embryo production for microinjection. One-cell embryos for microinjection were obtained using standard procedures as previously described in our laboratory 22 . Briefly, cumulus oocytes complexes (COCs) were retrieved from slaughterhouse ovaries of wild-type sheep. ...
Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.
... Ritchie et al. have applied a lentiviral transduction protocol with eGFP marker gene for both zona-free and split embryos and reported potential possibilities for the application of this approach in small ruminants (Ritchie et al., 2005;Ritchie et al., 2009). Perivitelline space injection of lentiviral vectors that carry eGFP marker genes has shown efficient expression of green Crispo et al., 2015b). Despite these results, using a 2A peptide-based tricistronic lentiviral vector for the expression of three fluorescent protein genes subjected to hypermethylation and silenced the expression of transgenes in transgenic sheep (Tian et al., 2013). ...
Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the generation of genetically modified sheep and goats. Further assisted tools have emerged to enhance the efficiency of genetic modification and to simplify the generation of genetically modified founders. These tools include sperm-mediated gene transfer, viral vectors, RNA interference, recombinases, transposons, and endonucleases. Of these tools, the four classes of site-specific endonucleases (meganucleases, ZFNs, TALENs, and CRISPRs) have attracted wide attention due to their DNA double-strand break-inducing role, which enable desired DNA modifications based on the stimulation of native cellular DNA repair mechanisms. Currently, CRISPR systems dominate the field of genome editing. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor. These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases. This review highlights the advances of genome engineering in sheep and goats over the past four decades with particular emphasis on the application of CRISPR/Cas9 systems.
... RhoD participated in the MAPK signaling pathway and various cell processes such as cell diff erentiation, cytokines production, and apoptosis. HSP10 participates in cell proliferation apoptosis, infl ammatory immune response and reproductive processes [9][10][11][12][13][14] . ...
... The IVEP was conducted using the previously described protocol (Crispo et al., 2014;Menchaca et al., 2016b). Briefly, for in vitro maturation, Grade 1 and 2 COCs were incubated for 24 h in drops of maturation medium (TCM 199 supplemented with 10% estrous sheep serum (ESS), 10 μg/ml FSH, 10 μg/ml LH, 100 μM cysteamine, 50 UI/ml and 50 μg/ml of penicillin and streptomycin, respectively) in a humidified atmosphere containing 5% CO 2 at a temperature of 39°C. ...
