Generation of biologically active retro-genes upon interaction of mouse spermatozoa with exogenous DNA

Summary: 
This study on mice shows how mRNA from ie mRNA vaccines can be incorporated in the sperm production cells, spermatozoas giving rise to DNA/Gene modifications in the next generation on mice. In this case a gene coding for a protein called EGFP was introduced in the next generation of mice.
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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 
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 plasmid harboring 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 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.
Fig. 1. PCR amplification of spliced EGFP reverse-transcribed copies in sperm cells after incubation with pBSKS-EGFP-INT DNA. A : Map of the vector containing the EGFP gene interrupted by a g globin intron in opposite orientation. The CMV early promoter and the TK poly(A) signal (pA) are indicated. SD and SA: splicing donor and splicing acceptor sites. Arrows indicate the oligonucleotide pairs, flanking the splicing sites, used for amplification. B : DNA was extracted and amplified from supernatants ( lanes 1–3 ) and nuclei 
 
TABLE 1 . PCR Screening of EGFP cDNA Sequences in F0 Pre-implantation Embryos, Fetuses, and Adult Mice
 
Fig. 2. PCR amplification of spliced EGFP reverse-transcribed copies in embryos obtained in IVF assays using pBSKS-EGFP-INT DNA-incubated spermatozoa. A: Lanes 1-7: two-cell embryos, lanes 8 and 9: EGFP-INT and EGFP control amplification, respectively. B: Lanes 1-5: four-cell stage embryos; lanes 6-9: morulae; lanes 10-11: blastocysts; lanes 12 and 13: EGFP-INT and EGFP control amplification, respectively. C: Lanes 1-2: 14 days fetuses; lanes 3-8: adults 20 days after birth; lanes 9 and 10: EGFP-INT and EGFP control amplification, respectively.
 
Fig. 3. Age-dependent changes in the pattern of EGFP-containing cDNAs in founder animals. Parallel PCR and Southern blot assays were carried out using DNA samples extracted from the tails of six animals of the same litter at 20, 40, and 60 days after birth. Numbers on the left hand side indicate the size of PCR amplification products of the unspliced (1,243 bp) and spliced (342) EGFP sequence. On the right hand side panels, the arrows indicate the hybridization signal of the Htf9/RanBP1 genomic probe used for Southern blot as a single-copy gene marker. M: lambda/HindIII DNA size marker. 
 
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