Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer

2005 ◽  
Vol 72 (1) ◽  
pp. 68-76 ◽  
Author(s):  
Nicole L. Webster ◽  
Monica Forni ◽  
Maria Laura Bacci ◽  
Roberto Giovannoni ◽  
Riccardo Razzini ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Proteins ◽  
Transgenic Pigs

scholarly journals Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 259-272 ◽  
Author(s):  
Francisco A García-Vázquez ◽  
Salvador Ruiz ◽  
Carmen Matás ◽  
M José Izquierdo-Rico ◽  
Luis A Grullón ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Reactive Oxygen Species Generation ◽  
Reca Protein ◽  
Transgenic Animals ◽  
Membrane Lipid ◽  
Transgenic Pigs ◽  
Exogenous Dna

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

scholarly journals Sperm-mediated gene transfer

2006 ◽  
Vol 18 (2) ◽  
pp. 19 ◽  
Author(s):  
Marialuisa Lavitrano ◽  
Marco Busnelli ◽  
Maria Grazia Cerrito ◽  
Roberto Giovannoni ◽  
Stefano Manzini ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Small Animal ◽  
Ease Of Use ◽  
Large Animal ◽  
Transgenic Pigs ◽  
Exogenous Dna ◽  
Large Animal Models

Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.

Poly-l-lysine-Based Molecular Conjugate Vectors: A High Efficiency Gene Transfer System for Human Progenitor and Leukemia Cells

2001 ◽  
Vol 321 (2) ◽  
pp. 129-136 ◽  
Author(s):  
Paul Schwarzenberger ◽  
Weitao Huang ◽  
Peter Oliver ◽  
Chris Theodossiou ◽  
Tolu Osidipe ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Leukemia Cells ◽  
Transfer System ◽  
Gene Transfer System ◽  
Molecular Conjugate

Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells

1982 ◽  
Vol 2 (6) ◽  
pp. 607-616
Author(s):  
Masahiro Ishiura ◽  
Susumu Hirose ◽  
Tsuyoshi Uchida ◽  
Yoshio Hamada ◽  
Yoshiaki Suzuki ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Gene Transfer ◽  
Mammalian Cells ◽  
Phage Particle ◽  
High Efficiency ◽  
Linear Increase ◽  
Recombinant Phage ◽  
Optimal Values ◽  
Simplex Virus

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk − cells to the TK + phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25°C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10 5 phage particles per 10 6 cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 × 10 7 phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10 10 phage particles per dish, did not significantly affect the number of transformants.

scholarly journals High-efficiency gene transfer and expression in normal human hematopoietic cells with retrovirus vectors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 811-814 ◽  
Author(s):  
P Laneuville ◽  
W Chang ◽  
S Kamel-Reid ◽  
AA Fauser ◽  
JE Dick
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
High Efficiency ◽  
Bone Marrow Cells ◽  
Fibroblast Cell ◽  
Retroviral Vectors ◽  
Semisolid Medium ◽  
Bacterial Gene

Abstract Retroviral vectors containing the selectable bacterial gene for G418 resistance (neo) were used to demonstrate gene transfer into primary human bone-marrow progenitor cells. To obtain populations of cells in which a high proportion of cells were expressing the neo gene, several important modifications were made to earlier procedures. Cells from normal donors were infected in vitro, were exposed to high concentrations of G418 for two days in liquid culture to enrich for cells expressing the neo gene, and were plated in semisolid medium. Gene transfer and expression were detected in colonies arising from progenitors of granulocyte-macrophage and erythroid lineages. Survival curves indicated that a high proportion of progenitor cells, approaching 100%, were G418 resistant. Furthermore, addition of growth factors contained in 5637-conditioned medium to the bone marrow improved the recovery of G418-resistant progenitors twofold to threefold. In addition to these biological measurements of gene expression in progenitor cells, significant levels of neo-specific RNA, similar to the levels of RNA expression in the virus-producing fibroblast cell line, were detected in the bone marrow cells after preselection. These results demonstrate that retrovirus vectors can be used successfully to transfer genes at high efficiency into progenitor cells in the human blood-forming system.

61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 148 ◽  
Author(s):  
J. Li ◽  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
...  
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Transgenic Pig ◽  
Transgenic Pigs ◽  
Uv Illumination ◽  
Polar Bodies ◽  
Transgenic Embryos ◽  
Handmade Cloning

Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P < 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P < 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells. Table 1.Comparison of two enucleation methods for the production of transgenic pig embryos

scholarly journals Kinetics of Recombinant Adeno-Associated Virus-Mediated Gene Transfer

2000 ◽  
Vol 74 (8) ◽  
pp. 3555-3565 ◽  
Author(s):  
Ajay K. Malik ◽  
Paul E. Monahan ◽  
David L. Allen ◽  
Bing-Guan Chen ◽  
R. Jude Samulski ◽  
...  
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Factor Ix ◽  
Lag Phase ◽  
Cmv Promoter ◽  
Adeno Associated Virus ◽  
Primary Myoblasts ◽  
Human Factor Ix ◽  
Hmw Dna ◽  
Kinetics Of

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to β-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 μg of hFIX/106 cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.

High efficiency gene transfer in rainbow trout (Salmo gairdneri Rich.) by microinjection into egg cytoplasm

Aquaculture ◽  
1986 ◽  
Vol 51 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Daniel Chourrout ◽  
Rene Guyomard ◽  
Louis-Marie Houdebine
Keyword(s):  
Rainbow Trout ◽  
Gene Transfer ◽  
High Efficiency ◽  
Salmo Gairdneri
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