Birth of cloned calves from vitrified-warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning.

The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2±2.2% vs 41.7±1.7% and 39.1±2.1%, respectively; P<0.01). The total cell number of BNF-derived blastocysts was significantly higher (P<0.01) than that of BFF-derived blastocysts, which, in turn, was higher (P<0.01) than that of BAF-derived blastocysts. Following transfer of vitrified-warmed blastocysts to recipients, no pregnancy was obtained with fresh (n=8) or vitrified-warmed (n=18) BAF-derived blastocysts, whereas transfer of fresh BNF- (n=53) and BFF-derived (n=32) blastocysts resulted in four and three pregnancies, respectively, which aborted within 90 days of gestation. The transfer of vitrified-warmed BNF-derived blastocysts (n=39) resulted in the live birth of a calf weighing 41kg, which is now 23 months old and has no apparent abnormality, whereas the transfer of vitrified-warmed BFF-derived blastocysts (n=18) resulted in one live birth of a calf that died within 6h. These results demonstrate that cloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

Genome editing of avian species: implications for animal use and welfare.

Avian species are used as model systems in research and have contributed to ground-breaking concepts in developmental biology, immunology, genetics, virology, cancer and cell biology. The chicken in particular is an important research model and an agricultural animal as a major contributor to animal protein resources for the global population. The development of genome editing methods, including CRISPR/Cas9, to mediate germline engineering of the avian genome will have important applications in biomedical, agricultural and biotechnological activities. Notably, these precise genome editing tools have the potential to enhance avian health and productivity by identifying and validating beneficial genetic variants in bird populations. Here, we present a concise description of the existing methods and current applications of the genome editing tools in bird species, focused on chickens, with attention on animal use and welfare issues for each of the techniques presented.

IL-2 in the tumor microenvironment is necessary for Wiskott-Aldrich syndrome protein deficient NK cells to respond to tumors in vivo.

To kill target cells, natural killer (NK) cells organize signaling from activating and inhibitory receptors to form a lytic synapse. Wiskott-Aldrich syndrome (WAS) patients have loss-of-function mutations in the actin regulator WASp and suffer from immunodeficiency with increased risk to develop lymphoreticular malignancies. NK cells from WAS patients fail to form lytic synapses, however, the functional outcome in vivo remains unknown. Here, we show that WASp KO NK cells had decreased capacity to degranulate and produce IFNγ upon NKp46 stimulation and this was associated with reduced capacity to kill MHC class I-deficient hematopoietic grafts. Pre-treatment of WASp KO NK cells with IL-2 ex vivo restored degranulation, IFNγ production, and killing of MHC class I negative hematopoietic grafts. Moreover, WASp KO mice controlled growth of A20 lymphoma cells that naturally produced IL-2. WASp KO NK cells showed increased expression of DNAM-1, LAG-3, and KLRG1, all receptors associated with cellular exhaustion and NK cell memory. NK cells isolated from WAS patient spleen cells showed increased expression of DNAM-1 and had low to negative expression of CD56, a phenotype associated with NK cells exhaustion. Finally, in a cohort of neuroblastoma patients we identified a strong correlation between WASp, IL-2, and patient survival.

Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA.

Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator-like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2-3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.

Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

Effect of cytoplasmic volume on developmental competence of buffalo (Bubalus bubalis) embryos produced through hand-made cloning.

This study examined the effects of cytoplasmic volume on the developmental competence of hand-made cloned buffalo embryos. Two different cell types, that is, buffalo fetal fibroblast (BFF) and buffalo embryonic stem (ES) cell-like cells were taken as donor cell and fused with one, two, or three demicytoplasts to generate embryos with decreased, normal (control), and increased cytoplasmic volume. Using BFF as a nuclear donor, the cleavage rate was similar in all the groups (p > 0.05), but the blastocysts rate was significantly lower (p < 0.05) for embryos generated with decreased cytoplasmic volume. Using ES cell-like cells, the cleavage and blastocyst rate with increased cytoplasmic volume was significantly higher (p < 0.05) compared that with reduced cytoplasmic volume. Blastocysts produced from embryos having increased cytoplasmic volume had significantly higher (p < 0.05) cell number than normal (control) embryos in both BFF and ES cell-like cells groups. Pregnancies were established in all the groups except for the embryos reconstructed with decreased cytoplasmic volume. The pregnancy rate was almost double for embryos reconstructed using increased cytoplasmic volume compared to that with the controls. Most of the pregnancies aborted in the first trimester and one live calf was delivered through Caesarean, which died 4 h after birth.