The use of lentiviral vectors to obtain transgenic rats (original) (raw)
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Efficient transgenic rat production by a lentiviral vector
American Journal of Physiology-Heart and Circulatory Physiology, 2007
A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken β-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit β-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and ∼100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F1population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference fo...
Proceedings of the National Academy of Sciences, 2002
The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies. E mbryonic stem (ES) cells are derived from early mammalian embryos and display characteristics of totipotency, i.e., after transfer to a suitable in vivo environment they contribute to the primary germ layers (ectoderm, endoderm, and mesoderm) and populate the germline of mice (1, 2). ES cells can be propagated in an undifferentiated state and genetically manipulated in vitro. Thus, transgenic animals can be generated by introducing foreign genes into ES cells, followed by transplantation of the ES cells into embryos and germ-line transmission.
1998
Lentiviral vectors are promising gene delivery tools capable of transducing a variety of dividing and non-dividing cells, including pluripotent stem cells which are refractory for transduction by murine retroviruses. Although there is a growing debate on the safety of lentiviral vectors for gene transfer, in particular for those derived from human immunodeficiency viruses, type one (HIV -1) and type two (HIV-2), these vectors are envisioned to possess several advantages . Importantly, they can be utilized not only for transducing specific target cells or for in vivo gene therapy of HIV infection and acquired immunodeficiency syndrome (AIDS), but also in a pseudotype recombinant form can be used for different target cells including neurological and cancer cell s. For HIV-2, the most compelling advantages are: (i) its reduced ability to recombine with resident HIV -1 genome; (ii) its ability to induce in recipients antibodies which can be distinguished from host immune response to HIV...
Molecular therapy : the journal of the American Society of Gene Therapy, 2001
Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer a...
2014
Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human �1-antitrypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 � 10 8 transducing units (T.U.)/mouse) had 81 % of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5‘-bromo-2‘-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expre...
Blood, 2002
High-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)-based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% ؎ 6.0%) and spleen (24% ؎ 3%). Most transduced hepatocytes and nonhepatocytes were nondivid-ing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II-positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo.
Germline Transmission and Tissue-Specific Expression of Transgenes Delivered by Lentiviral Vectors
Science, 2002
Single-cell mouse embryos were infected in vitro with recombinant lentiviral vectors to generate transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter . Eighty percent of founder mice carried at least one copy of the transgene, and 90% of these expressed GFP at high levels. Progeny inherited the transgene(s) and displayed green fluorescence. Mice generated using lentiviral vectors with muscle-specific and T lymphocyte–specific promoters expressed high levels of GFP only in the appropriate cell types. We have also generated transgenic rats that express GFP at high levels, suggesting that this technique can be used to produce other transgenic animal species.
Biomedicine & Pharmacotherapy, 2020
Lentiviral vectors (LVs) have provided an efficient way to integrate our gene of interest into eukaryote cells. Human immunodeficiency virus (HIV)-derived LVs have been vastly studied to become an invaluable asset in gene delivery. This abled LVs to be used in both research laboratories and gene therapy. Pseudotyping HIV-1 based LVs, abled it to transduce different types of cells, especially hematopoietic stem cells. A wide range of tropism, plus to the ability to integrate genes into target cells, made LVs an armamentarium in gene therapy. The third and fourth generations of self-inactivating LVs are being used to achieve safe gene therapy. Not only advanced methods enabled the clinical-grade LV production on a large scale, but also considerably heightened transduction efficiency. One of which is microfluidic systems that revolutionized gene delivery approaches. Since gene therapy using LVs attracted lots of attention to itself, we provided a brief review of LV structure and life-cycle along with methods for improving both LV production and transduction. Also, we mentioned some of their utilization in immunotherapy and gene therapy.