Generation of biologically active retro-genes upon interaction of mouse spermatozoa with exogenous DNA.
Mature spermatozoa of most animal species can spontaneously take up foreign DNA molecules which can be delivered to embryos upon fertilization. Following this procedure, transgenic animals of various species have been generated. We recently discovered a reverse transcriptase (RT) activity in mouse spermatozoa that can reverse-transcribe exogenous RNA molecules into cDNA copies. These cDNA copies are transferred to embryos at fertilization, mosaic propagated as non-integrated structures in tissues of founder individuals and further transmitted to F1 progeny. Reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals. To learn more about this mechanism and further characterize the reverse transcription step, we have now incubated spermatozoa with a plasmid harboring a green fluorescent protein (EGFP) retrotransposition cassette interrupted by an intron in the opposite orientation to the EGFP gene. We found that reverse-transcribed spliced EGFP DNA sequences are generated in sperm cells and transmitted to embryos in IVF assays. After implantation in foster mothers, embryos developed into mice that expressed EGFP in the blood vessel endothelia of a variety of organs. The EGFP-encoding cDNA sequences were detected in positive tissues as extrachromosomal mosaic-propagated structures, maintained in low-copy number (<1 copy/genome), and mosaic transmitted from founders to the F1 progeny. These results indicate that an efficient machinery is present in mature spermatozoa, which can transcribe, splice, and reverse-transcribe exogenous DNA molecules. This mechanism is implicated in the genesis and non-Mendelian propagation of new genetic information besides that contained in chromosomes.
Pittoggi
Carmine
C
Istituto Superiore di Sanità (ISS), Rome, Italy.
Beraldi
Rosanna
R
Sciamanna
Ilaria
I
Barberi
Laura
L
Giordano
Roberto
R
Magnano
Anna Rosa
AR
Torosantucci
Liliana
L
Pescarmona
Edoardo
E
Spadafora
Corrado
C
D000818:Animals; D001692:Biological Transport; D018076:DNA, Complementary / Q000032:analysis / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000737:chemistry / Q000378:metabolism*; D005333:Fetus / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007438:Introns; D008297:Male; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013094:Spermatozoa / Q000201:enzymology* / Q000378:metabolism; D014018:Tissue Distribution; D014158:Transcription, Genetic
D018076:DNA, Complementary; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012194:RNA-Directed DNA Polymerase