Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes.
Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely.
Larochelle
Nancy
N
Department of Neurology Friedrich-Baur-Institute, Ludwig-Maximilians-Universität München, Munich, Germany. nancy.larochelle@mcgill.ca
Stucka
Rolf
R
Rieger
Norman
N
Schermelleh
Lothar
L
Schiedner
Gudrun
G
Kochanek
Stefan
S
Wolf
Eckhard
E
Lochmüller
Hanns
H
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols; D002945:Cisplatin; D004622:Embryo, Mammalian / Q000166:cytology / Q000821:virology*; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007069:Ifosfamide; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D016685:Mitomycin; D009865:Oocytes / Q000821:virology*; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics / Q000502:physiology; D016662:Virus Integration
D049452:Green Fluorescent Proteins; D016685:Mitomycin; D002945:Cisplatin; D007069:Ifosfamide
Nat Biotechnol. 1996 Aug;14(8):982-5
9631035
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4407-11
8183921
Gene Ther. 1997 May;4(5):465-72
9274724
Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1401-5
7877990
Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7482-7
10840055
Nature. 1991 Aug 29;352(6338):815-8
1881437
Biol Reprod. 2002 Oct;67(4):1172-9
12297533
Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5731-6
8650161
Biol Reprod. 1997 Jan;56(1):119-24
9002640
Trends Genet. 1997 Aug;13(8):335-40
9260521
Life Sci. 1997;61(9):891-7
9284082
Cell. 1993 Apr 23;73(2):309-19
8477447
Gene Ther. 2003 Apr;10(7):580-4
12646863
Mol Ther. 2001 Dec;4(6):603-13
11735345
J Biol Chem. 2000 Dec 1;275(48):37915-21
10954710
J Virol. 1983 Sep;47(3):631-6
6620464
Gene Ther. 2000 Jan;7(1):31-4
10680013
Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3854-8
7731995
Science. 1997 Feb 28;275(5304):1320-3
9036860
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13565-70
8942974
Gene. 2009 Jan 1;428(1-2):20-4
18976699
Fertil Steril. 2008 May;89(5 Suppl):1448-54
17624347
J Virol. 1999 Jul;73(7):6141-6
10364373
Science. 1990 Mar 23;247(4949 Pt 1):1465-8
1690918
Hum Gene Ther. 2008 Oct;19(10):1000-8
18788904
Gene Ther. 1994 Sep;1(5):338-40
7584100
Mol Reprod Dev. 1995 Nov;42(3):291-7
8579842
EMBO J. 1982;1(9):1101-4
6329727
Curr Gene Ther. 2002 May;2(2):135-44
12109211
Mol Ther. 2000 Mar;1(3):263-74
10933942
Nat Genet. 1998 Feb;18(2):180-3
9462752
Curr Top Microbiol Immunol. 1995;199 ( Pt 2):109-37
7555065
Hum Gene Ther. 1998 Sep 20;9(14):2135-42
9759939
Proc Natl Acad Sci U S A. 2005 Sep 20;102(38):13628-33
16174752
J Gene Med. 2008 Nov;10(11):1176-89
18773501
J Clin Invest. 1998 May 15;101(10):2119-28
9593768
Hum Mol Genet. 1994 Apr;3(4):579-84
8069302
Mol Ther. 2001 Apr;3(4):557-64
11319918
Curr Top Microbiol Immunol. 1982;101:127-94
6303701
J Virol. 1996 Jun;70(6):3788-96
8648714
Hum Gene Ther. 2000 Aug 10;11(12):1705-12
10954904
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1383-8
11818536
Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3352-6
9096397