Murine <i>PGK</i>‐1 promoter drives widespread but not uniform expression in transgenic mice

McBurney, Michael W.; Staines, William; Boekelheide, Kim; Parry, David; Jardine, Karen; Pickavance, Lucy

doi:10.1002/aja.1002000403

Cited by 55 publications

(30 citation statements)

References 28 publications

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“…(iii) To achieve transgene expression not only in ES cells but also in differentiated derivatives and tissues of adult animals, we chose the CAG promoter (23), which contains a modified chicken ␤-actin promoter and enhancer sequences from the cytomegalovirus, and the promoter of the PGK. Both promoters have been shown to be ubiquitously active in the mouse (24,25). Three lentiviral vectors were designed (Fig.…”

Section: Resultsmentioning

confidence: 99%

Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

Pfeifer

Ikawa

Dayn

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

486

339

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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.The first reports of genetic manipulation of ES cells demonstrated that vectors derived from retroviruses can infect ES cells and that the integrated virus (provirus) is transmitted through the germline (3, 4). Furthermore, it was shown that retroviral vectors are able to infect preimplantation embryos, giving rise to transgenic animals that transmit the proviral DNA to offspring (5-8). However, further analysis revealed that both infected ES cells and preimplantation embryos lack significant provirus transcription. Two major mechanisms have been identified for retrovirus silencing (see references in ref. 9): trans-acting factors that bind to the viral promoters in the long terminal repeats (LTRs) and methylation of the integrated retroviral genome and flanking host DNA sequences. According to the organization of their genome (for review see ref. 10), one can distinguish simple retroviruses, such as the prototypic murine leukemia virus, from complex retroviruses like the lentiviruses. HIV type 1 (HIV-1) is one of the best-studied complex retroviruses and has the ability to infect nondividing cells presumably by import of the viral DNA through the nuclear pore and subsequent integration into the host genome (references in ref. 11). Vectors derived from lentiviruses can transduce a broad spectrum of terminally differentiated, nondividing cells, as well as, hematopoietic stem cells of multiple mammalian species (references in ref. 11).In this communication, we show that (i) unlike traditional oncoretroviral vectors, expression of transgenes introduced by lentiviral vectors into murine or human ES cells is not silenced.(ii) Transgene expression is not ''shut off'' during differentiation, and the transgene is expressed in multiple tissues of chimeric animals generated by transfer of lentivector-transduced ES cells in blastocysts. (iii) Germ-line transmission of transgenes introduced into ES cells by lentiviral vectors and (iv) preimplantation embryos at morula stage can be successfully transduced with lentiviral vectors, and the resulting progeny express the transgene. We therefore conclude that lentiviral vectors will be excellent tools for generating transgenic animals. Materials and MethodsVirus Production. LV-green fluorescent protein (GFP) was constructed by cloning the CAG promoter into the ClaI and BamHI sites of the vector LV-pGFP (12), thereby replacing the phosphoglycerate kinase (PGK) promoter. LV-Lac was cloned by introducing the LacZ-woodchuck hepatitis virus frag...

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Section: Resultsmentioning

confidence: 99%

Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

Pfeifer

Ikawa

Dayn

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

486

339

View full text Add to dashboard Cite

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“…54,56,[58][59][60] Given the recently demonstrated ability of the Pgk1 promoter to enhance minimal CMV activity in a bidirectional configuration, as well as the hypothesized chromatin insulating properties of dual, divergently transcribed promoters, we postulated that this promoter architecture would increase CMV transcription and prevent variegation in ES and XEN cells. 27,29,36,37 In addition, previous reports comparing promoter activity within ES and hematopoietic progenitor cells indicated that EEF1A1 was the strongest promoter tested.…”

Section: Bidirectional Expression Cassette Designsmentioning

confidence: 99%

A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells

Golding

Mann

2011

Gene Ther

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The two main challenges facing retroviral transgenesis are variable expression and epigenetic silencing. Although modern lentiviral vectors incorporate several elements to increase transgene expression and reduce position effect variegation and silencing, therapeutic research in stem cells, as well as production of transgenic animals, is still hampered by these two key problems. On the basis of recent studies demonstrating the chromatin insulating properties of divergent promoters, we sought to develop a bidirectional lentiviral vector with which to conduct RNA interference (RNAi)-based genetic screens in embryonic and extraembryonic stem cells. To this end, we designed and tested a series of synthetic bidirectional promoters, combining the mouse phosphoglycerate kinase 1 (Pgk1) promoter with other strong mammalian and viral promoters. Here, we demonstrate that a back-to-back configuration of the mouse Pgk1 and human eukaryotic translation elongation factor 1 alpha 1 promoters provided a substantive increase in both transgene expression and RNAi-based transcript depletion as compared with previous designs and other promoter combinations. Using this vector, we were able to achieve stable and robust depletion of a transfected luciferase reporter, as well as an endogenous non-coding RNA. The described constructs are an improved transgene delivery system capable of conducting RNAi screens in stem cells at single copy.

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“…This has been shown for several oncogenes retrovirally transduced into rat NEC grafts, which continue to proliferate in vivo after grafting. 34 The internal Pgk-1 promoter utilized in our study is active in embryonic and postnatal neural tissues 35 and the pM48 retrovirus construct with the internal Pgk-1 promoter has been shown to be less susceptible to rapid downregulation of retrovirus expression in vivo than LTR-based retrovirus constructs. 22 According to a popular hypothesis, 3 medulloblastomas originate from remnants of the external cerebellar granule cell layer (EGL).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of Pax5 is not sufficient for neoplastic transformation of mouse neuroectoderm

et al. 2001

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The developmental control genes of the Pax family are essential for brain development. Several Pax genes are also involved in chromosomal translocations causing malignancies in humans, and Pax5 expression is deregulated in medulloblastomas. We have investigated whether Pax5 can induce tumors in the developing mouse brain. Primary mouse embryonic neuroectodermal cells were retrovirally transduced with mouse Pax5 and transplanted into the brain of syngeneic host mice. No tumors developed in 36 transplants after one year, and there were no alterations in the differentiation pattern of the neural transplants. We then generated transgenic mice expressing human Pax5 under control of the Engrailed-2 promoter, which is expressed in the cerebellar external granule cell layer and in medulloblastomas. Sustained expression was achieved in the cerebellum of transgenic animals throughout lifetime. Expression levels were similar to those observed in human medulloblastomas. Again, cerebellar morphogenesis was undisturbed, and no tumors arose. These results strongly argue against a dominant transforming activity of PAX5 in NEC and in cerebellar granule cell precursors of mice, and underline the restricted tissue-specificity of PAX5 related oncogenesis.

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Murine PGK‐1 promoter drives widespread but not uniform expression in transgenic mice

Cited by 55 publications

References 28 publications

Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells

Overexpression of Pax5 is not sufficient for neoplastic transformation of mouse neuroectoderm

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