Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations

Ultrasound-induced sonoporation with the aid of magnetic nanoparticles

Targeted gene and drug delivery has untapped potential for the treatment of a wide range of pathological conditions. The recent advancements in ultrasound contrast agents (UCAs) including their ability to target regions of interest (ROI) with high specificity makes them ideal for a range of both in vitro and in vivo studies. This work investigates gene expression enhancement by a novel combination of nanosized magnetic particles (MagTag®) and cationic UCAs (cUCAs). The cUCAs have been studied alone and in combination with the MATra-A reagent (Iba Life Sciences, Goettingen, Germany), that uses activated magnetic fields for the delivery of nucleic acids into the cell nucleus

A case study on cryopreservation of African sheep semen for the Red Maasai, Dorper breeds and their crosses

The objective of this study was to examine the effect of different concentrations of trehalose and raffinose in Tris-citric acid-glucose (TCG) egg yolk extender for the sperm freezability of Red Maasai sheep, another African breed- the Dorper and their crosses. The results showed that Red Maasai, Dorper and their crosses sperm could be cryopreserved by extending with 0.075 to 0.01 M trehalose or raffinose-TCG egg yolk extender. We conclude that the cryopreservation of currently under threat Red Maasai sheep due to uncontrolled exotic breeds sperm makes it possible to establish a gene bank for the storage of animal genetic resources for endangered species, under the management of the International Livestock Research Institute for future genetic options in an uncertain world

Human adipose stem cell and ASC-derived cardiac progenitor cellular therapy improves outcomes in a murine model of myocardial infarction

Philip MC Davy,1 Kevin D Lye,2,3 Juanita Mathews,1 Jesse B Owens,1 Alice Y Chow,1 Livingston Wong,2 Stefan Moisyadi,1 Richard C Allsopp1 1Institute for Biogenesis Research, 2John A. Burns School of Medicine, University of Hawaii at Mānoa, 3Tissue Genesis, Inc., Honolulu, HI, USA Background: Adipose tissue is an abundant and potent source of adult stem cells for transplant therapy. In this study, we present our findings on the potential application of adipose-derived stem cells (ASCs) as well as induced cardiac-like progenitors (iCPs) derived from ASCs for the treatment of myocardial infarction. Methods and results: Human bone marrow (BM)-derived stem cells, ASCs, and iCPs generated from ASCs using three defined cardiac lineage transcription factors were assessed in an immune-compromised mouse myocardial infarction model. Analysis of iCP prior to transplant confirmed changes in gene and protein expression consistent with a cardiac phenotype. Endpoint analysis was performed 1 month posttransplant. Significantly increased endpoint fractional shortening, as well as reduction in the infarct area at risk, was observed in recipients of iCPs as compared to the other recipient cohorts. Both recipients of iCPs and ASCs presented higher myocardial capillary densities than either recipients of BM-derived stem cells or the control cohort. Furthermore, mice receiving iCPs had a significantly higher cardiac retention of transplanted cells than all other groups. Conclusion: Overall, iCPs generated from ASCs outperform BM-derived stem cells and ASCs in facilitating recovery from induced myocardial infarction in mice. Keywords: adipose stem cells, myocardial infarction, cellular reprogramming, cellular therapy, piggyBac, induced cardiac-like progenitor

Early fetal development of nuclear transfer bovine embryos generated from fibroblasts genetically modified by piggybac transposition

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117-8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19184-19189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey´s test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0±17.8 v. 100.0±16.1 and 2.8±0.8 respectively, n=4-7; P<0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development.Fil: Alessio, Ana Paula. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fili, Alejandro. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Forcato, Diego Oscar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Olmos Nicotra, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alustiza, Fabrisio Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Rodriguez, Natalia Evelin. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Sampaio, R. V.. Universidade de Sao Paulo; BrasilFil: Sangalli, J.. Universidade de Sao Paulo; BrasilFil: Bressan, F.. Universidade de Sao Paulo; BrasilFil: Fantinato-Neto, P.. Universidade de Sao Paulo; BrasilFil: Meirelles, F.. Universidade de Sao Paulo; BrasilFil: Owens, J.. John A. Burns School Of Medicine; Estados UnidosFil: Moisyadi, S.. John A. Burns School Of Medicine; Estados UnidosFil: Kues, W.A.. Friedrich-Loeffler-Institut; AlemaniaFil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin