Insights on bovine genetic engineering and cloning

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT

Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development.

Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3-6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3-6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3-6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3-6 mm follicles) in embryos. Interestingly, the addition of EVs from 3-6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3-6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus)

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such `labour-intensive technology`

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), SP, Brazil[06/58536-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), SP, Brazil[05/59694-5

Primer sequences of genes analyzed by real-time PCR.

<p>Primer sequences of genes analyzed by real-time PCR.</p

Global DNA methylation and hydroxymethylation levels in blastocysts after supplementation with small-extracellular vesicles (EVs).

<p>(A) Global DNA methylation levels in D7 blastocysts. (B) Global DNA hydroxymethylation levels in D7 blastocysts. (C) Confocal images of embryos that underwent different treatments during oocyte maturation and embryo development (40x objective). Different capital letters (A and B, A’ and B’ or A” and B”) indicate differences (p<0.05) among IVM groups within a given IVC group. Different small letters (a and b, a’ and b’, or a” and b”) indicate differences (p<0.05) among IVC groups within a given IVM group. Control (normal FCS), EVsfree-FCS (EVs-depleted FCS), EVs3-6mm (EVs from 3–6 mm in size follicles). Data are presented as mean ± s.e.m.</p

Characterization of small-extracellular vesicles (EVs) from 3–6 mm ovarian follicles.

<p>(A) The size of the EVs isolated from a pooled follicular fluid was evaluated by blockage of electrical current analysis, which demonstrated the presence of small size vesicles between 30-200nm. Different colors represent different replicates. (B) Transmission electron microscopy of isolated EVs demonstrated the presence of a homogenous population of small vesicles at 80kV (Scale bar 500nm). (C) EVs-RNA size distribution according to electropherogram of RNA molecules present in EVs from follicular fluid demonstrated the absence of 18s and 28s bands, as well as the presence of small RNA molecules. (D) Immunoblotting analysis of proteins in EVs and follicular cells. Herein we identified the presence of ALIX and CD63 two EVs markers, present in follicular fluid (FF), FF without EVs (FF-EV), follicular cells (FC), and EVs, isolated from 3-6mm ovarian follicles. We also identified the presence of PABP, an RNA binding protein, in FF, FF without EVs, follicular cells, and EVs. ACTB, a structural protein, was determined in the preparations, and demonstrated not to be enriched in EVs. Additionally, we verified the presence of Cytochrome C (CYT C), a mitochondrial protein, which serves as a negative control for cell contamination. EVs = extracellular vesicles <200 nm; ALIX = Programmed cell death 6-interacting protein; CD63 = CD63 molecule; PABP = Polyadenylate-binding protein 1; ACTB = Beta-actin; CYT C = Cytochrome c-1.</p

In vitro embryo developmental rates.

<p>(A) Blastocyst rates following small-extracellular vesicles (EVs) treatment during oocyte in vitro maturation (IVM) and embryo culture (IVC). Results are based on five independent in vitro production (IVP) routines in total ~979 cumulus-oocyte-complexes (COCs) (EVsfree-FCS = 318, EVs3-6mm = 318, EVsPreOv = 343). EVsfree-FCS (EVs-depleted FCS), EVs3-6mm (EVs from 3–6 mm in size follicles) and EVsPreOv (EVs from pre-ovulatory follicles). Different letters indicate statistical difference P<0.05.</p

The small-extracellular vesicles (EVs) uptake by an embryo in in vitro culture.

<p>(A) Blastocysts stained with DAPI (nuclear staining) with a 40x magnification. (B) Blastocysts stained with DAPI (nuclear staining) with a 40x magnification. C) Blastocysts exposed to PKH-67 labeled follicular fluid EVs during embryo culture (D1-D7) presenting a punctuated staining due to labeled small-extracellular vesicles, with a 40x magnification. D) Blastocysts exposed to PKH67 labeled PBS 1x during embryo culture (D1-D7) presenting a non-specific green staining with a 40x magnification. Blue = DAPI (nuclear staining); Green dots = PKH67 labeled small-extracellular vesicles (arrow).</p