CSIRO Publishing blank image blank image blank image blank imageBooksblank image blank image blank image blank imageJournalsblank image blank image blank image blank imageAbout Usblank image blank image blank image blank imageShopping Cartblank image blank image blank image You are here: Journals > Reproduction, Fertility and Development   
Reproduction, Fertility and Development
Journal Banner
  Vertebrate Reproductive Science & Technology
 
blank image Search
 
blank image blank image
blank image
 
  Advanced Search
   

Journal Home
About the Journal
Editorial Board
Contacts
Content
Online Early
Current Issue
Just Accepted
All Issues
Special Issues
Research Fronts
Sample Issue
For Authors
General Information
Instructions to Authors
Submit Article
Open Access
For Referees
Referee Guidelines
Review Article
Annual Referee Index
For Subscribers
Subscription Prices
Customer Service
Print Publication Dates

blue arrow e-Alerts
blank image
Subscribe to our email Early Alert or RSS feeds for the latest journal papers.

red arrow Connect with us
blank image
facebook twitter youtube

red arrow Connect with SRB
blank image
facebook TwitterIcon

Affiliated Societies

RFD is the official journal of the International Embryo Transfer Society and the Society for Reproductive Biology.


 

Article << Previous     |     Next >>   Contents Vol 22(1)

431 GENERATION OF CLONED CD55-CD39 TRANSGENIC α1,3-GALACTOSYLTRANSFERASE DEPLETED (GAL-/-) PIGLETS

A. Perota A, D. Brunetti A, B. Charreau D, M. Chatelais D, I. Lagutina A, G. Lazzari A, I. Anegon D, D. H. Sachs A, E. Cozzi E, F. Lucchini C, C. Galli A

A Laboratorio di Tecnologie della Riproduzione, AVANTEA srl, Cremona, Italy;
B Università di Bologna, Dipartimento Clinico Veterinario, Ozzano Emilia, Italy;
C Università Cattolica del Sacro Cuore, Centro Ricerche Biotecnologiche, Cremona, Italy;
D Institut de Transplantation Et de Recherche en Transplantation CHU, INSERM UMR 643, Nantes, France;
E Direzione Sanitaria, Ospedale generale di Padova, Padova, Italy;
F Massachusetts General Hospital and Harvard Medical School, Transplantation Biology Research Center, Boston, MA USA
 
 Export Citation
 Print
  


Abstract

Success in xenotransplantation relies on engineering of the pig genome to express human transgenes, such as CD55/CD39, that can control coagulation and inflammation to prolong the graft survival of a 1,3-galactosyltransferase depleted (Gal-/-) pig organs in nonhuman primates and then able to bypass the hyperacute rejection. The aim of our work was to produce Gal-/- piglets overexpressing CD55/CD39. In experiment (Exp.) 1 exploiting 2 ubiquitous expression vectors (pCAGGS-CD55 and pCAGGS-CD39), we transfected immortalized porcine kidney cells (PK15) with CD55 and CD39 using Nucleofector (Amaxa Biosystems, Cologne, Germany) and selected 5 cell colonies each (PK15-CD55 and PK15-CD39) that were expanded and analyzed by RT-PCR, immunohistochemistry (IHC) and Western blot (WB). The monoclonal antibodies IA10 for hCD55 and BU61 for hCD39 were used. Transgenic transcription was confirmed by Northern blot (NB) using digoxigenin-labeled probes. In Exp. 2, a neonatal pig Gal-/- fibroblast line was co-transfected by Nucleofector using 2 ubiquitous expression vectors (hEF-CD55 and pCAGGS-CD39) for the expression of CD55 and CD39. Colonies were analyzed by RT-PCR and IHC only, because of the limited number of cells available. Cells from one colony with a high level of CD55/CD39 expression according to IHC were used for nuclear transfer into enucleated oocytes. Day 5 compact morula/blastocyst (n = 144) were transplanted in 2 synchronized sows. Porcine aortic endothelial cells (PAEC) and fibroblasts derived from 2 stillborn piglets were analysed with IHC, NB, and WB. The expression level of transgenes from both experiments was compared with human umbilical vein endothelial cells (HUVEC) by fluorescence-activated cell sorting (FACS), using IA10, BRIC110, IH4, 2G2, and MEM-118 antibodies for hCD55 and TU66 for hCD39. In Exp. 1, RT-PCR showed CD55 mRNA expression in 3 out of 5 (2, 15, 24) PK15-CD55 colonies. A high level of CD55 expression was confirmed only in colony 24 by IHC, NB, WB, and FACS. Low expression level in colony 2 revealed by FACS was not detected by IHC, indicating that FACS analysis is more accurate to quantify the level of expression. All PK15-CD39 colonies were positive according to RT-PCR and IHC. Only one colony PK15-CD39 was further analyzed by NB and WB and confirmed positive. In Exp. 2, IHC, NB, WB, and FACS analyses of fibroblasts and PAEC derived from both cloned piglets confirmed the high level of CD39 expression detected by IHC in donor cells used for nuclear transfer. However, strong CD55 expression detected by IHC was not confirmed by NB analyses and, by FACS, was lower than in HUVEC cells. In conclusion, we produced cloned CD55-CD39 transgenic Gal-/- piglets with a high level of CD39 expression but the expression level of CD55 was lower than in HUVEC cells. We found that although IHC is the method of choice in preliminary screening, it is not sufficiently quantitative when only a few cells for each clone are available. Thus, IHC needs to be complemented with additional methods (e.g. WB, FACS, real-time RT-PCR) to obtain complete evaluation of the expression pattern of transgenes before nuclear transfer experiments.

   
    
Legal & Privacy | Contact Us | Help

CSIRO

© CSIRO 1996-2014