Repositorio
Institucional

Felmer, Ricardo

Sanchez-Villalba, Esther#Arias, Maria Elena#Loren, Pia#Fuentes, Fernanda#Pereyra-Bonnet, Federico#Salamone, Daniel

Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O

ELSEVIER SCIENCE BV

ANIMAL REPRODUCTION SCIENCE

en

The ICSI-sperm mediated gene transfer (ICSI-SMGT) has been used to produce transgenic mice with high efficiency, however, the efficiency of this technique in farm animals is still less than desirable. Pretreatment of sperm with membrane destabilizing agents can improve the efficiency of ICSI in cattle. The objective of the present study was to evaluate streptolysin-O (SLO) as a novel treatment to permeabilize the bovine sperm membrane and assess its effect on efficiency of generating transgenic embryos by ICSI-SMGT. First, there was evaluation of the plasma membrane integrity (SYBR/PI), acrosome membrane integrity (PNA/FITC), DNA damage (TUNEL) and binding capacity of exogenous DNA (Nick Translation) in bull sperm treated with SLO. Subsequently, there was assessment of embryonic development and the efficiency in generating transgenic embryos with enhanced expression of the gene for green fluorescent protein (EGFP). Results indicate that SLO efficiently permeabilizes the plasma and acrosome membranes of bull spermatozoa and increases binding of exogenous DNA mostly to the post-acrosomal region and tail without greatly affecting the integrity of the DNA. Furthermore, treatment of bull spermatozoa with SLO prior to the injection of oocytes by ICSI-SMGT significantly increased the rate of embryo expression of the EGFP gene. Future experiments are still needed to determine the effect of this treatment on the development and tansgene expression in fetuses and animals produced by ICSI-SMGT.

Artículo original

This research was supported by FONDECYT Grants 11130724, and 1160467 CONICYT, Chile and Directorate of Research Grants DI17-5006 and DI16-2001, Universidad de La Frontera. E. Sanchez and P. Loren were supported by a Doctoral scholarship by CONICYT, Government of Chile.

Esta investigación fue financiada por las Becas FONDECYT 11130724 y 1160467 de CONICYT, Chile, y las Becas de Investigación DI17-5006 y DI16-2001 de la Dirección de la Universidad de La Frontera. E. Sánchez y P. Loren recibieron una beca de doctorado de CONICYT, Gobierno de Chile.

10.1016/j.anireprosci.2018.07.005

pdf

SLO# SMGT

http://dx.doi.org/10.1016/j.anireprosci.2018.07.005

Agriculture, Dairy & Animal Science# Reproductive Biology# Veterinary Sciences

SLO# SMGT

Biotecnología agrícola

Tecnología de modificación genética

Ciencia Animal y lechería

WOS:000447110900015

Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O

hdl.handle.net/11336/91412

Artículo original

version publicada

ELSEVIER SCIENCE BV

ANIMAL REPRODUCTION SCIENCE

Agricultura, Lácteos y Ciencia Animal# Biología Reproductiva# Ciencias Veterinarias

0378-4320

en

UFRO DI17-5006#UFRO DI16-2001#ANID FONDECYT 11130724#ANID 1160467

ANID FONDECYT 11130724

ANID FONDECYT 1160467

UFRO DI17-5006

UFRO DI16-2001

This research was supported by FONDECYT Grants 11130724, and 1160467 CONICYT, Chile and Directorate of Research Grants DI17-5006 and DI16-2001, Universidad de La Frontera. E. Sanchez and P. Loren were supported by a Doctoral scholarship by CONICYT, Government of Chile.

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