E-News and Reviews:
Paper review: Transgenesis by adenovirus-mediated gene transfer
"Transgenesis by adenovirus-mediated gene transfer into zona-free eggs"; Nature Biotechnology Vol.14 (August 1996), pp982-985. Tohru Tsukui, Yumi Kanegae, Izumu Saito and Yutaka Toyoda This paper explores the possibility of generating transgenic animals by the use of a method with 2 major advantages over that of the accepted microinjection procedure. They propose the use of replication-defective adenoviruses to achieve this goal, a method that has been tested in the past as a possible delivery system or vehicle for somatic gene therapy. In order to determine that infection has occurred and a functional transgene has been incorporated into the genome, they used Lac Z as a reporter and assayed embryos using a B-galactosidase staining test. It should be noted that no attempts to utilize this type of delivery system have been previously reported - this is believed to be due to previous reports that viral integration into the host genome was rare. Previous work carried out by this lab had demonstrated successful infection of zona-free mouse eggs and subsequent expression in cleaving pre-implantation embryos, however Lac Z expression was undetectable at embryonic day 13.5 . This initial construct coupled a replication-defective adenovirus containing the Lac Z gene under the control of SRalpha promoter. In order to clarify the possibility of viral integration and exogenous gene expression, a further construction was made, with the Lac Z gene tagged to a nuclear localization signal controlled by a CAG promoter (chicken B-actin and CMV-1E). The effectiveness of the infection procedure on embryos with or without a zona pellucida was tested by the use of X-gal staining for the presence of B-galactosidase activity. Stain could be seen clearly in the nuclei of 2-cell stage embryos that had developed from the infected eggs without a zona after 24 hours; it was conspicuously absent from the eggs where the zona had not been removed prior to "infection". To evaluate the procedure further, infected eggs were cultured to blastocyst stage and implanted into pseudopregnant foster mothers by standard procedures. This group obtained 3 founder animals from 27 live born animals (11.1%) as determined by Southern blot analysis of tail biopsies from these potential founder animals. Examination of the restriction patterns from each of the 3 founders was highly suggestive of random integration of the adenoviral DNA; furthermore the restriction patterns (minus only the terminal fragments) were shown to be almost identical to the control viral DNA suggesting that in each case the entire construction was integrated intact. Indeed, the restriction patterns suggest that only a single copy was integrated, as no more than 2 novel-sized junction fragments were detected in each case. The function of the Lac Z promoter was determined by tissue assay using X-gal staining (tissue obtained by ear punch). 2 of the 3 founders showed B-galactosidase activity, 1 did not. It is suggested that this failure is due to a lack of integration of the entire Lac Z expression unit. The group demonstrated germline transmission in all the founder animals to their F1 progeny, and in the 2 lines where the Lac Z reporter was functional the transgenic F1s demonstrated the presence of B-galactosidase activity by X-gal staining while their non-transgenic siblings did not. The conclusion was that the adenovirus-mediated transgene was stably transmitted to the next generation, and that this transmission followed expected Mendelian pattern of inheritance without transgene rearrangement. The distribution of Lac Z expression at 12 months old in the F1 mice was strong in the heart, muscle and brain, with low to moderate levels in the spleen, intestine, kidney and testis. Additionally, microscopic analysis of postnatal F1 mice (frozen sections) showed extensive nuclear-targeted B-galactosidase activity in the intercostaris muscle and rib, and in the heart. Additionally, high expression levels were noted in the hippocampus and cerebellum. In the discussion the virtues of the single copy integration are extolled, with the authors' belief that this may be due to the terminal proteins of the adenoviral genome inhibiting the formation of tandem clusters. Also concluded was that integration of the adenovirus occurred prior to or at the time of DNA synthesis at the 1-cell stage, thereby allowing subsequent stable germline transmission. The absence of the E1A gene (one of 2 removed to render the virus replication-defective) is also thought to help the adenoviral DNA exist in a stable state - the many rearrangements in cell lines transformed with wild-type adenovirus is cited here.
Experimental Protocol Infection of pronuclear eggs with the Adenoviral (Ad) - Lac Z vector B6C3F1 hybrid mice were used. IVF was carried out to collect fertilized eggs*. 1 to 2 hours after insemination the zona pellucidas were removed, and the zona-free eggs infected with the Ad-Lac Z construct at a concentration of 1x10 8 pfu/ml. Virus stock was added into droplets of WM-EDTA medium each containing 40-80 zona-free eggs under mineral oil, and co-incubated for 6 hours at 37C in 5% CO2 in air. Eggs were subsequently washed 4x in WM-EDTA medium and cultured individually in 5ul droplets of WM-EDTA for up to 96 hours post insemination before being transferred into pseudopregnant mothers at the blastocyst stage (ICRs used). * Hoshi, M. and Toyoda, Y. "1985 "Effect of EDTA on the preimplantation development of mouse embryos fertilized in vitro" Jpn. J. Zootech. Sci. 56:pp931-937 The main advantages to this procedure seem to be:
- Not required to invest in costly microinjection equipment or have the associated high level of technical expertise to generate transgenic animals.
- Can sidestep the multiple copy numbers often produced during standard microinjection as a result of concatamer formation - particularly relevant if the target gene is thought to have a dosage effect.
Although an interesting and informative paper from a technical standpoint, I must reserve judgement on the efficiency of this technique in transgenic production. The impression from reading the experimental protocol is that several droplets containing 40-80 eggs each are prepared for each day's effort, yet 27 live born animals are reported. More informative would be discussion of the mortality of embryos that undergo each successive step, allowing evaluation of each procedure (coculture, culture to blasts, implantation, pregnancy rate, numbers implanted per foster mother, etc). Also, more hard numbers are needed to assess what an achievable rate of transgenesis is for this procedure - an n=27 is not as informative as I would like. Nevertheless, the authors have utilized and described a technique that has considerable potential for development and may prove a valuable tool for transgenic research.
