Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer.
Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins.
Webster
Nicole L
NL
The Austin Research Institute, Studley Road, Heidelberg, Victoria, Australia.
Forni
Monica
M
Bacci
Maria Laura
ML
Giovannoni
Roberto
R
Razzini
Riccardo
R
Fantinati
Paolo
P
Zannoni
Augusta
A
Fusetti
Lisa
L
Dalprà
Leda
L
Bianco
Maria Rosaria
MR
Papa
Michele
M
Seren
Eraldo
E
Sandrin
Mauro S
MS
Mc Kenzie
Ian F C
IF
Lavitrano
Marialuisa
M
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics*; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D051379:Mice; D009028:Morula / Q000166:cytology / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*; D013552:Swine / Q000196:embryology / Q000235:genetics*
D049452:Green Fluorescent Proteins