325 Generation of a Cloned Green Fluorescent Protein (Gfp) Expressing Transgenic Sheep for Muscle Stem Cell Graft Experiments

Abstract

Sheep are a good model for cardiopathy and surgery training in medical studies because organ volumes are similar to humans. Grafting of stem cells collected from skeletal muscles is a major area of research into treatments for heart failure. To establish an efficient protocol it is first necessary to follow the fate of grafted cells in an animal model. The aim of the project was to obtain 2 cloned sheep of the same genetic background, 1 conventional and 1 expressing green fluorescent protein (GFP), to be used for graft experiments. First, chimeric transgenic fetuses were generated by transduction of 8-cell stage embryos with a lentivirus expressing GFP under the EF1a human ubiquitous promoter. A large dilution of the lentivirus solution was used so that only some cells were transduced. Chimeric transgenic embryos were examined for GFP expression and transferred to recipients at the blastocyst stage. At 50 days of pregnancy, 8 fetuses were obtained. Three showed stable but mosaic expression of GFP in some tissues, as expected. The proportion of green cells ranged from 20 to 80% between fetuses. To make sure that low-level expression was not overlooked in GFP-negative fetuses, skin cells from each of the 8 fetuses were cultured for 10 days to isolate green from white cell colonies. This step confirmed the negative signal in 5 fetuses, but also led to the elimination of 1 positive fetus whose cells tended to switch off the GFP signal. Only 1 fetus yielded a good enough ratio of white to green cell colonies to enable the freezing of cells, which were subsequently used in 4 NT experiments. In total, 42 blastocysts were transferred to 20 recipients, of which 4 reached late pregnancy. A GFP-positive cloned fetus was delivered by C-section 4 days before term and required no intensive care. This animal is now over 6 months old and clinically normal. Expression of GFP in skin is stable and readily visualised with specialised GFP glasses. Global expression of GFP in all tissues will be followed in an F1 generation to avoid risk of contamination after biopsies in this first precious animal. Weekly ultrasound examination revealed the onset of fetal suffering (abdominal fluid accumulation, reduced heart rate, and fetal movements) in the last week before term in 3 other fetuses. These did not survive despite emergency C-section and intensive neonatal care. Fetal anomalies were similar to those observed in bovine NT. Gross placental abnormalities, however, were not present. None of the postmortem observations could be attributed to lentivirus integration as they were similar to those seen in nontransgenic cloned animals. Experiments are now proceeding to generate a normal white cloned sheep by NT using frozen nontransgenic cells from the same fetus. This will allow generation of sheep with the same genetic background that can be used to develop muscle stem cell grafting protocols. The authors thank M. Bonneau, CRII, for help in C-section, and J. P. Albert, J. Massoneau, and S. Rotg for animal care.

Pregnancy and Neonatal Care of SCNT Animals

“…At C-section, one fetus was stillborn, while the other died shortly after birth despite intensive resuscitation with the help of an experienced human neonatologist. The third lamb required almost no special care (Boulanger et al, 2010), and is still alive and healthy.…”