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a Department of Obstetrics and Gynecology, Mt. Sinai Medical Center, New York, New York 10029
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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conceptus, developmental biology, early development, embryo, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another attractive strategy for germ line gene transfer is infection of gametes or embryos with recombinant viruses. This technique requires no special equipment or difficult micromanipulation, it does not require visualization of subcellular compartments such as the pronucleus, it can insert genes with minimal disruption of host DNA structure, and it has the potential for efficient gene transfer. In mice, the discovery that retroviruses can infect embryos [15] was later used to produce transgenic mice with recombinant retroviruses [16]. More recently, retroviral vectors have been shown to infect mature oocytes [17, 18] and have been used to produce transgenic monkeys [18]. Retroviruses can also be introduced into male germ line cells via infection in vitro followed by cell transplantation into the seminiferous tubule [19]. However, retroviruses have major limitations as germ line gene transfer vectors. They have a relatively low cloning capacity, about 9 kilobases (kb), and their expression in adult animals has often been low [16]. For these reasons, a new vector system would be highly advantageous.
In this context, the report that adenovirus gene transfer vectors can insert into gametes or embryos is of substantial interest. Farre et al. [20] reported that exposure of porcine sperm to adenovirus vectors followed by in vitro fertilization led to apparent integration of the adenovirus DNA into the genome. There were some inconsistencies in these data, including that the presence of adenovirus DNA could not be confirmed with Southern blots despite apparent widespread positivity of reverse transcription-polymerase chain reaction (PCR) tests for expression of the reporter gene, bacterial ß-galactosidase (lacZ), which was cloned into the vector. Moreover, our own efforts to reproduce these findings in mice were unsuccessful [21]. Another study in which fertilized mouse ova were exposed to an adenovirus vector that expressed lacZ and then transferred reported that 3 of 27 pups were transgenic for the vector DNA and transmitted that DNA through the germ line [22]. This finding, if it is readily reproducible, has the potential to significantly augment the extant germ line gene transfer armamentarium. Adenovirus vectors can be produced in high titers, can infect a wide variety of cell types, and can accommodate more than 20 kb of exogenous DNA [23]. The potential importance of this report of transgenesis after embryo infection with adenoviruses [22] to the advance of germ line gene transfer technology has accordingly been emphasized [24]. However, few experiments have been performed to follow up this finding.
In the present study, we have examined in more detail the potential for adenovirus vectors to introduce foreign DNA into the mouse germ line. The susceptibility of embryos to infection and the toxicity of vectors was examined. We have also transferred embryos after exposure to such a vector and tested large numbers of fetuses and newborns for the presence of foreign DNA. Results indicate that adenovirus vectors cannot be readily used for germ line gene insertion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An adenovirus, ß-galactosidase (Adß-gal) vector in which the lacZ gene is driven by the Rous sarcoma virus (RSV) promoter [21] was obtained from the Vector Core, Institute of Gene Therapy and Molecular Medicine, Mt. Sinai Medical Center. Another vector with the lacZ gene driven by the human cytomegalovirus (CMV) promoter was obtained from the Institute for Human Gene Therapy of the University of Pennsylvania Health System. Both vectors were replication-defective, with deletions of E1 and E3. The viral titers of the RSV-lacZ preparation was 7 x 1012 particles/ml (p/ml) and 3 x 1011 infectious particles/ml (PFU/ml), and that of the CMV-lacZ vector was 5.2 x 1012 particles/ml and 5 x 1010 PFU/ml. These preparations were diluted to a concentration of 1 x 109 PFU/ml in KSOM [25] medium (MR-023-D; Specialty Media, Phillipsburg, NJ) and stored in 20-µl aliquots. Embryos were exposed to the vector by adding the 20 µl of stock to 1980 µl of KSOM to yield a final titer of 1 x 107 PFU/ml.
Mice
All experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Mt. Sinai School of Medicine. FVB/N and BDF1 mice were obtained from Taconic Farms, Germantown, NY. CD-1 mice were obtained from Charles River Laboratories, Wilmington, MA. Animals were maintained under conventional conditions in a 14L:10D cycle. Immature FVB/N females were superovulated by i.p. injection of 5 IU of pregnant mares serum (G4877; Sigma Chemical Company, St. Louis, MO) followed 48 h later by 5 IU of hCG (CG-10; Sigma), and placed with FVB/N males to mate. On the day following mating, embryos at the pronuclear stage were recovered by opening the oviducts in prewarmed KSOM supplemented with 2 mg/ml hyaluronidase (H-3884; Sigma) to remove the cumulus cells. Zygotes with visible pronuclei were then either exposed directly to adenovirus vector or cultured overnight for exposure to the vector at the two-cell stage.
To expose embryos to the adenovirus vectors, the zonae pellucidae were first removed by use of Tyrode buffer (T-1788; Sigma) supplemented with 1:250 vol/vol of concentrated HCl, which was gently applied to the oocytes with a mouth pipette [26]. Immediately after zona dissolution, the eggs were washed three times in fresh KSOM and placed in 2 ml of medium that contained 1 x 107 or 1 x 108 PFU/ml of the adenovirus vector. When two-cell embryos were exposed, the KSOM was supplemented with BSA (A-3311; Sigma) to a final concentration of 4 mg/ml. Preliminary work had demonstrated that the added BSA did not affect the efficiency of embryo infection by adenovirus (data not shown). BSA was added and the embryos were maintained on a shaking platform for the incubation period in order to reduce adherence of embryos to the culture dish surface, which was greatly increased by the presence of adenovirus in the culture medium. Once adhered to the surface, the embryos could not be removed for washing without causing lysis. After various exposure times the embryos were washed in three changes of medium. The washing entailed collection of the embryos, transfer in a minimal volume to 2 ml of fresh medium in a 33-mm tissue culture dish, and repeated aspiration. With each change of medium the transfer pipette was changed in order to minimize carryover of virus particles that had adhered to the inner surface of the pipette. Embryos were then cultured individually in 10-µl microdrops of KSOM medium under mineral oil equilibrated with 10% vol/vol of Earles balanced salt solution (E-2000; Sigma). For all experiments, controls for the effectiveness of the washing procedure were performed by culturing zona-free embryos not exposed to the vector in microdrops produced from the final wash medium. These embryos were later stained for LacZ activity. No positive staining was observed after washing by these methods.
LacZ Staining of Embryos
This was performed exactly as published previously [21]. Briefly, embryos were pooled in 10-µl microdrops of KSOM under mineral oil. Excess medium was drained with a mouth pipette and replaced with 1.25% glutaraldehyde in PBS. The draining and replacement procedure was repeated two additional times, after which the process was reversed in order to replace the glutaraldehyde with KSOM. Embryos were then collected and placed in organ culture dishes that contained 1 ml of LacZ staining solution prepared as follows: 5.5 ml H2O, 10 µl of 1 M MgCl2, 28 µl of 4 M NaCl, 333 µl of 1 M Hepes pH 7.3, 750 µl of 0.03 M potassium ferrocyanide, 750 µl of 0.03 M potassium ferricyanide, and 7 µl of saturated NaOH. To 1 ml of this mixture was added 26 µl of X-gal, 20 mg/ml dissolved in dimethyl formamide. This staining solution was prepared fresh immediately before each test, and the ferrocyanide and ferricyanide solutions were made fresh every 3 days. To minimize evaporation during staining, the sidewell of the organ culture dish was filled with 3 ml of distilled H2O. Embryos were stained either for 4 h or overnight at 37°C in a standard tissue culture incubator. Staining of experimental and positive controls was performed simultaneously using the same preparation of staining solution.
Embryo Transfer
On the day of one-cell embryo recovery, mature CD-1 females were mated to vasectomized BDF1 males and examined the following morning for vaginal plugs. After culture to the compacted morula/blastocyst stage (72 h after recovery of the one-cell embryos), females were anesthetized and the uterine horn was exposed through a dorsal incision. The uterine wall was pierced with a 25-gauge needle and the embryos were introduced through the needle track into the uterine lumen using a glass micropipette. Animals were allowed to carry pups to term for DNA analysis or were killed at 10–12 days of gestation for LacZ staining.
DNA Analysis
Newborns were reared to weaning and subjected to tail biopsy and DNA extraction according to the method of Laird et al. [27]. To perform PCR for the lacZ gene, the following primers were used: forward primer sequence, 5'-CAA ACT GGC AGA TGC CAC GGT TAC G-3'; reverse primer sequence, 5'-TAT GCA GCA ACG AGA CGT CAC GGA A-3'. Thirty-five cycles of PCR were performed after 5 min of denaturation at 94°C, and then 94°C for 0.5 min, 60°C for 0.5 min, and 72°C for 0.5 min. Products were visualized after electrophoresis in 3% MetaPhor agarose (BioWhittaker Molecular Applications, Rockland, ME). As a positive control for the PCR, 50 pg of a plasmid containing the lacZ gene was added to DNA from a normal animal. The band size expected for the lacZ gene was 385 base pairs (bp). PCR for the endogenous Cu/Zn superoxide dismutase gene was performed on duplicate samples to rule out the possibility that DNA isolates were not suitable for PCR. For these procedures, primers, reaction protocols, and gel analysis were performed exactly as previously published [28] except that FspI enzyme digestion of the reaction product was omitted.
For Southern hybridization, 15 µg of DNA was digested with appropriate restriction enzymes and subjected to electrophoresis in agarose and transfer to nylon membranes according to standard procedures. As a positive control, DNA extracted from the CMV-lacZ vector was added to DNA from a normal mouse at 50 pg/15 µg. For hybridization, radioactive probes were produced using a random priming kit obtained from Boehringer-Mannheim (Mannheim, Germany). Hybridizations were performed using the QuikHyb kit from Stratagene (La Jolla, CA) according the manufacturer's instructions. After washing at 65°C in 0.1 M NaCl, 1.5 mM sodium citrate pH 7.0, and 0.1% SDS, membranes were dried and exposed to x-ray film at -70°C for a suitable time.
LacZ Staining of Fetuses
Fetuses at 10–12 days of gestation were removed to 1.25% glutaraldehyde in PBS and fixed for 30 min at 4°C. They were then washed in PBS three times with 10-min incubations, and immersed in the same staining solution that was used for staining preimplantation embryos.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When zona-free one-cell mouse embryos were exposed to the RSV-lacZ adenovirus vector for 6 h at 108 PFU/ml, and were washed and cultured, minimal staining was observed at the morula stage (Fig. 1A). However, when the same vector preparation was exposed to two-cell embryos for 2 h at 1 x 107 PFU/ml, then washed and stained at the morula stage, marked LacZ staining was observed (Fig. 1B). Staining tests could not be performed when higher titers of virus were used because of embryotoxicity (see below). The staining of a subset of blastomeres in a small percentage of embryos was tentatively interpreted as indicating absence of viral receptor at the one-cell stage, with persistence of a small amount of virus, either passively attached to the cells or remaining in the medium after washing.
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Adenovirus Vectors Are Highly Toxic to Two-Celled Embryos, but Not to One-Celled Embryos
When one-cell embryos were exposed to 1 x 108 PFU/ml of the RSV-based vector for 6 h and cultured, 89% of the embryos reached the blastocyst stage. This rate of development was not significantly different from controls subjected to zona removal but not exposed to the virus (Table 1). However, when two-cell embryos were exposed for only 2 h, and washed and cultured, only 59% of them reached the blastocyst stage. This smaller number of normally developing embryos was highly statistically significant by chi-square analysis (Table 1). The absence of toxicity to one-cell embryos, in addition to the absence of reporter gene expression after challenge of one-cell embryos led us to conclude that functional adenovirus receptor does not appear until the two-cell stage. In order to maximize the opportunity to obtain germ line gene transfer after infection of two-cell embryos, we increased the titer of vector and attempted to obtain higher levels of infection without excessive loss of embryo viability. When two-cell embryos were exposed to 1 x 108 PFU for 2 h, none of the 76 embryos survived to the blastocyst stage (Table 1). When embryos were exposed at 1 x 107 PFU/ml for 6 h, blastocyst cavities were observed in 52% of embryos, but the blastocysts that formed exhibited a number of abnormalities that included delayed blastocyst formation and loss of blastomeres. Nonetheless, because of our interest in obtaining germ line integration, we attempted three embryo transfers with these blastocysts (30 embryos per transfer) and obtained no pregnancies. Developmental abnormalities were observed at 1 x 107 PFU/ml exposure for 2 h; these abnormalities were less severe, and we were able to obtain pregnancies after embryo transfer. Accordingly, we used this latter protocol in our attempts to produce transgenic mice.
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Further Studies to Evaluate Timing of Adenovirus Receptor Appearance
Our findings of differential toxicity and staining after exposure of one- and two-cell embryos to adenovirus vectors contrasts with a previous report [22] that exposure of embryos at the one-cell stage leads to high, uniform expression at the two-cell stage. We hypothesized that in the previous study, one-cell embryos were not completely washed, and residual virus remained until the embryos cleaved to the two-cell stage and produced a receptor. This postulate would demand that adenovirus vectors remain biologically active after overnight culture in mouse embryo medium, and that the virus could infect the two-cell embryo and produce LacZ enzyme before cleavage to the four-cell stage. This latter requirement would contrast with a previous study involving adenovirus that reported that 30 h is required for production of LacZ protein after exposure of embryos by subzonal insertion of the vector [29].
We accordingly performed an experiment that addressed both of these issues as follows. Adenovirus was placed in KSOM at 1 x 107 PFU/ml and allowed to remain in the medium overnight. The CMV-lacZ vector was used for these experiments because preliminary experiments had shown that the RSV vector did not express LacZ until the morula stage, whereas LacZ staining does occur at the two-cell stage when the CMV enhancer is employed [22]. At the time the vector was placed in culture, and fertilized eggs were recovered and also placed in culture. The following day, two-cell embryos were subjected to zona removal and were placed in the vector-containing medium. The embryos were allowed to incubate for 11 h, at which time the embryos, still at the two-cell stage, were stained for LacZ. As shown in Figure 2, heavy LacZ staining is apparent in nearly all embryos. These results show that adenovirus vectors can survive in mouse medium for at least 24 h, and further show that the adenovirus genome can be activated within 12 h of embryo infection, a time substantially shorter than was previously observed [29].
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To further assess the possibility that infection does not occur until after the two-cell stage is reached, we performed an experiment to determine the relative amounts of LacZ enzymatic activity in each of the blastomeres of two-cell embryos. We reasoned that infection at the one-cell stage would lead to an equal distribution of vector DNA, RNA, and LacZ enzyme molecules to the blastomeres of subsequent stages. On the other hand, infection of each blastomere at the two-cell stage could be asynchronous, and the amounts of LacZ enzymatic activity in each of the cells could differ. Our typical staining protocols involved incubation of the blastomeres overnight in the staining solution. With this procedure, differences in enzymatic activity would not be discernible because prolonged action of the LacZ enzyme saturates the cells with reaction product. Thus, cells with relatively low LacZ activity are just as heavily stained as those with high activity. To distinguish different levels of LacZ activity in blastomeres at the two-cell stage, we performed an experiment in which one-cell embryos were cultured in adenovirus overnight, and the resultant two-cell embryos were examined after 4 h of staining. As shown in Figure 3, different levels of LacZ activity are clearly present in each of the cells of several two-cell embryos. This finding provides further evidence that infection occurs after cleavage to the two-cell stage.
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Tests for Adenovirus DNA Integration in Embryos and Fetuses
Although several experiments indicated that adenovirus vectors do not infect embryos before the two-cell stage, it is still possible that integration after the first cleavage division could lead to production of transgenic mice with germ line gene insertion. We therefore tested newborns and fetuses for insertion of genes derived from the gene transfer vectors as described below.
Two-cell embryos were exposed to 1 x 107 PFU/ml of the CMV-lacZ vector for 2 h, and were washed and cultured to the compacted morula/blastocyst stage. We had previously observed that this infection protocol was capable of yielding high infection rates and vector gene expression in the embryo (see Fig. 1). Embryos that cleaved normally were then transferred to the uteri of pseudopregnant females and allowed to develop to term. After weaning, tail biopsies were performed followed by DNA extraction and PCR testing for lacZ gene sequences. Of 57 pups tested in this manner, one yielded a positive PCR signal. This animal's sample, with 36 other test samples, is shown in Figure 4 (sample 4). As this figure demonstrates, all other samples are negative, whereas PCR for the endogenous SOD1 gene, which is present as a single-copy gene, shows that all samples are of suitable quality for PCR (Fig. 4B).
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In order to investigate the possibility that this positive sample was due to contamination, a second tail biopsy was performed and the reaction was repeated. A positive signal was again detected, which indicates that adenovirus DNA was present in the animal. Unfortunately, this female died due to a traumatic event unrelated to the gene transfer before her progeny could be tested. However, to determine whether the animal was a genetic mosaic for the adenovirus DNA or whether the DNA was present in most or all cells, the tail DNA used for PCR was evaluated by Southern hybridization. We estimate that our Southern analyses are capable of detecting a copy of a target sequence in 1 in 10 cells. As shown in Figure 5, the tail sample was negative by Southern blotting after digestion of the sample with several enzymes. This finding indicates that if this animal was actually transgenic, it was a mosaic, with adenovirus DNA present in fewer than 1 in 10 cells.
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These findings led us to explore whether mosaic integration had occurred in other embryos but escaped detection because of the presence of DNA in only a subset of cells. To evaluate this question, embryos were exposed to the CMV-LacZ vector at the two-cell stage, cultured to the morula/blastocyst stage, and transferred into pseudopregnant females. To assure that infection was successful, a subset of the embryos were reserved for LacZ staining and not transferred. At 8–10 days of gestation, embryos were recovered and stained for LacZ. We reasoned that integration into a subset of cells in preimplantation or early postimplantation development would lead to "patches" of LacZ positive cells. These patches could easily be missed using PCR analysis if only a small biopsy were taken. On the other hand, failure of the virus to integrate would lead to the presence of fewer than 50 virions in the entire fetus, which we would expect to be undetectable by LacZ staining.
Thirty-seven embryos were evaluated in this way. Although both pre-embryo infection and staining were effective in all transfers evaluated, none of 37 embryos at <10–12 days of gestation exhibited any LacZ staining. In summary, two different tests for adenovirus integration, applied to 94 embryos, revealed a single positive PCR test that could not be confirmed by Southern hybridization.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When toxicity is balanced against low degrees of infection to produce viable embryos with relatively uniform and high expression of a reporter gene (Fig. 1B), embryo transfer yields a very low number of transgenic animals. We therefore conclude that unless significant modifications are made in adenovirus vectors, they will not serve as useful tools for germ line gene insertion.
When attempting to introduce a gene into the germ line, it is highly advantageous to employ an approach that leads to integration at the one-cell stage. Integration at later stages will perforce lead to mosaic integration and a commensurately reduced chance of obtaining germ line transmission. Using measures of toxicity and reporter gene expression, we have obtained data from three different experiments that provide compelling evidence that fully functional adenovirus receptor does not appear on mouse embryos until the two-cell stage. Adenovirus reporter gene activity (Fig. 1), and toxicity (Table 1) are both minimal after exposure of one-cell embryos, and substantial after exposure of two-cell embryos. Moreover, the pattern of LacZ staining of two-cell embryos and morulae is indicative of infection after the first cleavage division (Figs. 1 and 3). Further indirect but consistent evidence of infection after the first cleavage was the marked increase in adherence of infected two-cell embryos to the culture dish surface as compared with one-cell embryos. These surface characteristics are indicative of a qualitatively different interaction between the cells and the vector after the first cleavage.
We made attempts to visualize the adenovirus receptor by immunofluorescence. The receptor has two components—the coxsackie and adenovirus receptor (CAR), which is coupled with either the 
vß3 or vß5 integrin molecule [31]. We obtained an antibody to mouse CAR from J. Bergelson, Children's Hospital of Philadelphia, and attempted to stain two-cell and one-cell embryos by indirect immunofluorescence for comparison. Although two-cell embryos did stain more strongly, low levels of staining relative to background as well as variability in staining made it impossible to draw firm conclusions regarding the first appearance of CAR (data not shown). We also used a commercial phycoerythrin-conjugated antibody against v integrin (Pharmingen), but for similar reasons these experiments did not yield firm conclusions regarding the first appearance of these integrins on the embryo surface. A problem we encountered, which we believe extends to all such analyses, is that dramatic changes in embryo topography that occur as cleavage progresses make any but the most obvious differences in surface molecules difficult to demonstrate: whereas cell volumes progressively diminish, surface area and surface curvature increase. Assays for either RNA or protein by Northern or Western blotting are also inconclusive because they cannot localize the receptor to the cell surface.
Although results of three independent experiments all indicate that fully functional adenovirus receptor is not present on one-cell embryos, this conclusion has not been formally proven. Rare staining of morulae after exposure of one-cell embryos could be the result of the presence of very few receptors. Uneven staining of two-cell embryos could similarly be the result of infection at the one-cell stage with asynchronous development of mechanisms required to translocate the viral genome to the nucleus for activation of the reporter gene. However, although explanations can be invoked to explain these findings other than appearance of functional receptor after the first cleavage, these three independent experiments all yielded results that were consistent with this conclusion, and we therefore suggest that adenovirus receptor is either very rare or entirely absent at the one-cell stage.
As a control for effective washing of embryos after exposure to the vector, we grew zona-free embryos in the final wash medium. None of 86 embryos grown in this medium exhibited any LacZ staining. However, we invariably saw low levels of staining of one-cell embryos exposed to vector and then washed prior to the first cleavage (Fig. 1A). We speculate from these observations that one component of the bipartite adenovirus receptor, either CAR or integrin, is present on the surface of one-cell embryos, and that interaction with that component leads to low-affinity binding that impedes complete removal of vector by washing. Appearance of the other portion of the receptor after the first cleavage then leads to internalization of the residual vector, which results in low-level staining.
PCR analysis of 57 pups born after exposure of two-cell embryos to the adenovirus vector yielded a single positive sample, which could not be confirmed by Southern blotting (Figs. 3 and 4). Because two separate biopsies of the animal were positive, we believe that a small percentage of cells did in fact carry the adenovirus DNA. Although this animal could not be progeny tested, we believe it possible, though highly improbable, that germ line transmission of the DNA could have taken place. Because the vector was replication defective, with deletions of the early genes E1 and E3, which are required for replication, and because adenovirus cannot replicate in mouse cells [32], the only reasonable explanation for a positive PCR test is integration of the vector DNA in at least some cells. Adenovirus is known to integrate after infection in vitro [33], although the frequency of integration is very low (10-3 to 10-5 of infected cells). Our findings with mouse embryos are entirely consistent with these previous findings, and indicate that whereas integration into the germ line is possible after infection of two-cell embryos, it is not likely to occur with sufficient frequency to render these vectors efficient vehicles for introduction of genes into the germ line.
This conclusion is reinforced by our evaluation of 37 whole fetuses for LacZ staining after embryo infection and transfer. None of 37 midgestation embryos showed any evidence of integration, this despite the fact that the infection procedure was documented to be effective by staining of embryos that were not transferred, and confirmation that the staining reaction was effective. Because the CMV enhancer directed widespread expression of the lacZ gene in transgenic mice [22, 34], we believe these examinations constitute a sensitive test for mosaic integration. That no staining was seen is again consistent with rare or absent integration.
We conclude from these investigations that it may be possible to introduce genes into the germ line with adenovirus vectors. However, germ line integration is likely to be less frequent than with other methods because of the inability to introduce the vector at the one-cell stage, the toxicity of the virus, and the generally low frequency with which adenoviruses integrate. Our results contrast somewhat with a previous report of relatively frequent transgenic mouse production by infection of one-cell embryos with adenovirus vectors [22]. The differences between ours and the published findings may relate to the specific characteristics of the vector isolate. Each vector preparation has a unique ratio of infectious particles to noninfectious capsids that are capable of interacting with the receptor. Characteristics of the preparation could affect the relative toxicity of the vector to embryos. Given the necessity for balancing toxicity against suitable levels of infection, and the absence or severe paucity of functional receptor on the one-cell embryo, we believe the current generation of vectors is unsuitable for routine transgenic mouse production, and is probably also not ready for use in livestock. Further modifications of these vectors, which might include removal of more viral genes, introduction of sequences that favor integration, or modification of the envelope in such a way as to permit efficient infection at the one-cell stage, might in the future render these vectors highly useful for germ line gene transfer in mammals. Efforts in this direction have recently been published, and involved incorporation of retroviral long terminal repeats into the adenovirus construct [35]. This might increase integration frequency, although further modification of the viral proteins that would allow efficient infection of the one-cell embryo would also favor germ line integration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 4 January 2002.
First decision: 13 February 2002.
Accepted: 30 April 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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V integrins in adenovirus cell entry and gene delivery. Microbiol Mol Biol Rev 1999 63:725-733
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HindIII molecular weight markers is shown on the right