Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2–6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.     

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.     

Retroviral gene transfer inXenopus cell lines and embryos

Summary A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three diffeentXenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10⁸ cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the β-galactosidase gene into the neural tube lumen of 24-h embryos yielded β-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer inXenopus embryos and cell lines.     

DNA-mediated genetic transformation of mouse embryos and bone marrow--a review

In recent years, new gene transfer systems have been developed which allow molecularly cloned genetic material to be introduced into whole organisms. These systems include the microinjection of DNA into mammalian embryos, transfection of DNA into mouse bone marrow cells, and the infection of early embryos with retroviruses. Exogenous DNA appears to integrate randomly into the host genome. The production of transgenic mice by injection of DNA into mouse embryos has rapidly gained importance as an experimental tool for the study of gene regulation during development. Through this technique, recombinant molecules of any type can be introduced into one-celled embryos, and thus can be used to study development from its earliest stages. DNA sequences have been shown to integrate and transmit through the germ line to subsequent generations as mendelian traits. Transgenic mice carrying various gene constructs have been successfully exploited for the elucidation of factors which determine tissue specificity of gene expression as well as the level of gene control. Phenotypic changes related to expression of foreign genes have also been observed. This experimental approach thus promises to rapidly solve many of the heretofore most challenging problems in developmental genetics. Insertion of foreign genes has also made possible the creation of insertional mutants which manifest themselves most frequently as recessives. Such mutations can be readily studied at the molecular level by using the transferred material as a probe for recovery of the affected host sequence from genomic libraries. Many of these same problems have been addressed by introducing retroviral DNA into mouse embryos. Here, the sequences used for transfer have been limited to retroviral genes, but nonetheless these experiments have been profitably exploited for studies both of gene regulation and mutagenesis. Gene transfer systems are being developed allowing the experimenter to transfer DNA into bone marrow cells of mice, after which the recipient cells can be reintroduced into lethally irradiated histocompatible animals. This system has the advantage that selection can be applied during the gene transfer process such that the expression of the foreign material is assured. In addition, these experiments have created a model system for production of animals carrying a subpopulation of cells which is highly resistant to a toxic agent. This system has the potential for therapeutic application to man.     

Adenoviral vectors for gene transfer and therapy

Due to the very efficient nuclear entry mechanism of adenovirus and its low pathogenicity for humans, adenovirus-based vectors have become gene delivery vehicles that are widely used for transduction of different cell types, especially for quiescent, differentiated cells, in basic research, in gene therapy applications, and in vaccine development. As an important basis for their use as gene medicine, adenoviral vectors can be produced in high titers, they can transduce cells in vivo with transgenes of more than 30 kb, and they do not integrate into the host cell genome. Recent advances in the development of adenoviral vectors have brought considerable progress on issues like target cell specificity and tropism modification, long-term expression of the transgene, as well as immunogenicity and toxicity in vivo, and have suggested that the different generations of non-replicative and replicative vectors available today will each suit best for certain applications.     

Nanos3 maintains the germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathways

Cell death in the germ line is controlled by both positive and negative mechanisms that maintain the appropriate number of germ cells and that prevent the possible formation of germ cell tumors. In the mouse embryo, Steel/c-Kit signaling is required to prevent migrating primordial germ cells (PGCs) from undergoing Bax-dependent apoptosis. In our current study, we show that migrating PGCs also undergo apoptosis in Nanos3-null embryos. We assessed whether the Bax-dependent apoptotic pathway is responsible for this cell death by knocking out the Bax gene together with the Nanos3 gene. Differing from Steel-null embryos, however, the Bax elimination did not completely rescue PGC apoptosis in Nanos3-null embryos, and only a portion of the PGCs survived in the double knockout embryo. We further established a mouse line, Nanos3-Cre-pA, to undertake lineage analysis and our results indicate that most of the Nanos3-null PGCs die rather than differentiate into somatic cells, irrespective of the presence or absence of Bax. In addition, a small number of surviving PGCs in Nanos3/Bax-null mice are maintained and differentiate as male and female germ cells in the adult gonads. Our findings thus suggest that heterogeneity exists in the PGC populations and that Nanos3 maintains the germ cell lineage by suppressing both Bax-dependent and Bax-independent apoptotic pathways.     

Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.     

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.     

Replication-defective vectors of reticuloendotheliosis virus transduce exogenous genes into somatic stem cells of the unincubated chicken embryo.

Replication-defective vectors derived from reticuloendotheliosis virus were used to transduce exogenous genes into early somatic stem cells of the chicken embryo. One of these vectors transduced and expressed the chicken growth hormone coding sequence. The helper cell line, C3, was used to generate stocks of vector containing about 10(4) transducing units per ml. Injection of 5- to 20-microliters volumes of vector directly beneath the blastoderm of unincubated chicken embryos led to infection of somatic stem cells. Infected embryos and adults contained unrearranged integrated proviral DNAs. Embryos expressed the transduced chicken growth hormone gene and contained high levels of serum growth hormone. Blood, brain, muscle, testis, and semen contained from individuals injected as embryos contained vector DNA. Replication-defective vectors of the reticuloendotheliosis virus transduced exogenous genes into chicken embryonic stem cells in vivo.     

Site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the Cre/loxP-expression-switching system

As vectors, adenoviruses (Ads) have many attractive advantages for in vivo gene therapy. However, Ads do not usually integrate into the host genome and gene expression is, thus, transient. Adeno-associated virus (AAV) integrates into a specific locus (AAVS1) on the human host's chromosome 19, while conventional recombinant AAV (rAAV) vectors do not possess this property because such vectors lack the rep gene. AAV vectors carrying the rep gene do not have enough space for insertion of a transgene. We have constructed a hybrid adenovirus/adeno-associated virus (Ad/AAV) vector which has the advantages of both Ads and AAVs. Given that the rep gene products inhibit propagation of Ads, we used the Cre/loxP-expression-switching system to regulate the expression of the rep gene. The Ad/AAV vector easily propagates, can efficiently infect a broad range of cell types, and can integrate into a specific locus on host chromosomes.     

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.     

Transgenesis in chickens

The application of transgenic technology to domestic poultry offers an alternative means to conventional practice for improvement of this highly productive agricultural species. The hen's reproductive system has unique characteristics which have imposed limitations on the use of established methods for artificial gene transfer. In this article, we review the various strategies that have been adopted to overcome the problem. Target sites for gene insertion include the fertilized ovum, the blastodermal embryo in the unincubated egg, and the primordial germ cells. Notable success in obtaining somatic and germline transformation has been achieved with the use of retroviral vectors to infect the blastodermal embryo. Current attempts to introduce DNA directly into the genome, without resort to pathogen-derived vectors, are discussed.     

Infection by retroviral vectors outside of their host range in the presence of replication-defective adenovirus.

Retrovirus infection is normally limited to cells within a specific host range which express a cognate receptor that is recognized by the product of the env gene. We describe retrovirus infection of cells outside of their normal host range when the infection is performed in the presence of a replication-defective adenovirus (dl312). In the presence of adenovirus, several different ecotropic vectors are shown to infect human cell lines (HeLa and PLC/PRF), and a xenotropic vector is shown to infect murine cells (NIH 3T3). Infectivity is demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, selection with G418 for neomycin resistance, and PCR identification of the provirus in infected cells. Infectivity is quantitatively dependent upon both the concentration of adenovirus (10(6) to 10(8) PFU/ml) and the concentration of retrovirus. Infection requires the simultaneous presence of adenovirus in the retrovirus infection medium and is not stimulated by preincubation and removal of adenovirus from the cells before retrovirus infection. The presence of adenovirus is shown to enhance the uptake of fluorescently labeled retrovirus particles into cells outside of their normal host range, demonstrating that the adenovirus enhances viral entry into cells in the absence of the recognized cognate receptor. This observation suggests new opportunities for developing safe retroviral vectors for gene therapy and new mechanisms for the pathogenesis of retroviral disease.     

Production of adenovirus vector for gene therapy

The field of gene therapy is rapidly expanding with a major focus on the treatment of cancer. Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. However, the production of adenoviral vectors for gene therapy applications faces a number of challenges that limit the availability of high quality material at the early stages of research and development in the gene therapy field. Moreover, very few papers have been published on the subject and information on large-scale production methods are only available through specialized conference proceedings. This review outlines the problems associated with mass production of adenovirus vectors and describes research efforts by a number of groups who have contributed to optimize production methods. Better understanding of the adenovirus infection and replication kinetics as well as better understanding of complementing cell line physiology and metabolism greatly contributed to improving vector titers and volumetric productivity at higher cell densities. Also, the critical aspect of viral vector quantitation is discussed.     

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.     

Methods for construction of adenovirus vectors

Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

In vivo analysis of a new lacZ retrovirus vector suitable for cell lineage marking in avian and other species

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.     

High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebra fish

Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate