les4 - 5.4. Alternatives for pro-nucleus injection...

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5.4. Alternatives for pro-nucleus – injection pro-nucleus injection : some problems : 1) low yields (TG / n injected or transferred zygotes) 5-10 % in mouse max 1-4 % in rabbit, pig, cattle 2) technically not simple, often pro-nuclei hard to see 3) random integration (insertion mutagenesis/ mozaic expression) 4) concatamers (stability, reproducibility) 5) delayed integration (extra generation)
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Alternatives for pro-nucleus –injection intracytoplasmatic injection retroviral vectors spermatozoa as carrier nucleus transplantation locus-specific knock-in genetic manipulation male germ cells
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1. Intracytoplasmatic injection of DNA - faster compared to pro-nucleus inj. - developed for large DNA molecules - less technical demanding - interesting alternative major problem : degradation of DNA in cytoplasm mouse experiments with DNA + poly-L-lysine in zygotes - 13 % (TG/injected zygotes)= not bad - random integration/concatamers/delayed integration - technical simple - but very different from exp. to exp. (reproducibility )
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2. Retroviral vectors (alternative to pro-nucleus, mozaicism) - interesting alternative / high efficiëncy - but lot of work small inserts 5-6 kb still delayed integration (because most viruses infect dividing cells) BL-2 facility needed (bio-safety)
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1990 ies some breakthroughs in basic knowledge biology retroviruses - Infect dividing cells - integration is depending on destruction of nucleus-membrane (M phase) - infection of zygote or blastula leads always to delayed integration mozaicism - why not infecting oöcytes? cf. stay long in M-II phase (no nuclear membrane) cattle ovaries from slaughterhouse (PNAS 1998)
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oöcyte injection of retrovirus in perivitelline space, then IVF no delayed integration!! high yield!! High Troughput possible Polar body oocyt Zona pellucida Inj. In perivitel.
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2001: Science, 291, 309, 2001: first transgenic primate - GFP transgene (reporter gene) via replication-defective retroviral vector injection in perivitelline space + IVF by intra-cytoplasmatic sperm injection - 30 tranfers: 1 transgenic: ANDI: fluorescent cells/GFP mRNA - Germline? - Very interesting breakthrough
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2002: Science, 295, 868, 2002: use of Lentivirusses - Lentivirusses are retrovirusses - Infect deviding AND non-deviding cells - A priori problems: possible replication competent viruses random integration of active LTRs ”SIN” vectors with mutant LTRs: trcr. silent
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- HIV-SIN with GFP gene: 1. inj. in perivitel. space mouse zygotes : 76% of offspring Tg! 2. Zygotes: first remove ZP (Tyrod’s solution) + incubate in solution with virusses: all born pups Tg and germline transmission - very interesting: high effic./simple/cheap/less invasive - less problems with LCR (variable expression) - Also in rat system (difficult pronuclei) - disadvantage : max. 10 kb/ several integration places per zygote
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3. Direct gene transfer via spermatozoa - 1971 rabbit spermatozoa + SV40 DNA 16% of DNA was associated with spermatozoa, that were still fertile - 1989 start using this technique for TG
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les4 - 5.4. Alternatives for pro-nucleus injection...

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