Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system

Kodama, Daisuke; Nishimiya, Daisuke; Nishijima, Ken ichi; Okino, Yuuki; Inayoshi, Yujin; Kojima, Yasuhiro; Ono, Kosuke; Motono, Makoto; Miyake, Katsuhide; Kawabe, Yoshinori; Kyogoku, Kenji; Yamashita, Takashi; Kamihira, Masamichi; Iijima, Shinji

doi:10.1016/j.jbiosc.2011.10.006

Cited by 18 publications

(13 citation statements)

References 33 publications

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“…A stable tissue-specific expression of the recombinant gene in several generations of transgenic birds was observed when these DNA fragments were included in the gene constructs. However, the synthesis of the recombinant product was 20-50 times lower compared to the results obtained when using constitutive promoters [16]. It indicates the effect on the ovalbumin synthesis regulation system from several regulatory elements located both inside and next to or outside the structural gene.…”

Section: Introductionmentioning

confidence: 71%

Influence of Ovalbumin Gene Regulatory Elements on Tissue Specificity and Level of Transgene Expression

Волкова¹,

Fomin²,

Dotsev³

et al. 2016

S-h. biol.

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A b s t r a c tThe use of lentiviral vectors for the genetic modification of embryonic chicken cells is regarded as one of the promising methods for producing transgenic poultry. In this case, it is very important to determine the regulatory elements of the ovalbumin gene, providing tissue-specificity and high transgene expression in cells of chicken oviduct. The aim of this work was to study the effect of the intron sequences and promoter of ovalbumin gene on tissue specificity and level of the transgene expression. For this purpose constructs based on lentiviral vector pWpxl, containing eGFP marker gene under control of the modified ovalbumin gene regulatory elements were obtained. Vector pW2.8 included a chromosomal DNA fragment of 2.8 kb comprising a first exon, intron sequence and part of the second exon of the ovalbumin gene; vector pW1.2 -chromosomal DNA fragment of 1.2 kb comprising a promoter and ovalbumin gene sequence (without the intron and the first exon) to the transcription initiation point; vectors pW131, pW225, pW315 -chromosomal DNA fragment, similar to the fragment of 1.2 kb in pW1.2 vector in which promoter (80 bp) was replaced by highly structured sequence of the birds -actin gene promoter region of 131, 225 or 315 bp respectively. The control vector pWCAGgfp included constitutive hybrid regulatory element comprising human cytomegalovirus early gene enhancer and birds' -actin gene promoter (CAG). Primary culture cells of chick oviduct and human fibrosarcoma cells 293T (control) were used as target cells for transfection. Viral preparation was added after a monolayer of cells reached concentration of 1-3½10 7 CFU/ml. eGFP expression was determined by fluorimetry in 72 hours after transfection. Low level of expression of eGFP gene controlled by chromosomal fragment of 2.8 kb leader region of the ovalbumin gene was confirmed in vitro using culture of chicken oviduct cells and 293T cell line: in vector pW2.8 recombinant protein expression level was up to 25 times lower compared to pWCAGgfp vector with a constitutive promoter CAG. Yet the eGFP expression levels for pW1.2 and pW2.8 constructs were identical, indicating the absence of introns' influence on the expression level of the recombinant DNA using this regulatory element. When the ovalbumin gene promoter was replaced by highly structured elements of -actin constitutive gene promoter the increase in the expression of eGFP in 2-3 times was observed, as well as the increased expression occurred with lengthening of the promoter region of -actin gene (vectors pW131, pW225, pW315). The achieved levels of expression with the use of exogenous -actin gene promoter were comparable with expression levels controlled by a constitutive promoter CAG, however when the promoter part of the ovalbumin gene was replaced by exogenous promoter (gene -actin), the deregulation of tissue-specific expression of eGFP was observed, indicating that transcription with tissue-specific ovalbumin promoter gene can be modulated or activated with exogenous enhancers.

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Section: Introductionmentioning

confidence: 71%

Influence of Ovalbumin Gene Regulatory Elements on Tissue Specificity and Level of Transgene Expression

Волкова¹,

Fomin²,

Dotsev³

et al. 2016

S-h. biol.

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“…pLSi/CMVST6/ DAeGFP was constructed by inserting the expression cassette of chicken sialyltransferase 6 controlled by a CMV promoter into pLSi/DAeGFP. The expression vector of human erythropoietin (hEPO), in which basal expression induced by the truncated ovalbumin promoter was amplified in an automatic feed-forward manner by the TRE sequence and tTA transcription factor, was constructed by inserting the hEPO expression unit in a bicistronic manner by the internal ribosome entry site (IRES) into the MSCV-based vector (Kodama et al 2012). The vector backbone was changed to pSicoR, which was designated as pLSi/ OVA-TRE-hEPO-IRES-tTA/CMVeGFP-W.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Enhanced lentiviral vector production in 293FT cells expressing Siglec-9

et al. 2014

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Siglecs, sialic acid-recognizing Ig-superfamily lectins, regulate various aspects of immune responses, and have also been shown to induce the endocytosis of binding materials such as anti-Siglec antibodies or sialic acid-harboring bacteria. In this study, we demonstrated that the expression of Siglec-9 enhanced the transfection efficiency of several cell lines such as macrophage RAW264 and non-hematopoietic 293FT cells. We applied this finding to the production of a lentiviral vector in which cells were transfected simultaneously with multiple vectors, and achieved a twice increase in viral production levels. Furthermore, 293FT cells expressing lectin-defective Siglec-9 produced threeto seven-fold higher titer of viral vector compared with parental 293FT cells. These results suggest that Siglec-9 enhanced lentiviral vector production in a lectin-independent manner.

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“…In addition to this report, Kodama et al . () reported a novel system in which expression by the Tet‐regulating promoter can be amplified by a self‐feed forward mechanism using a hybrid promoter containing a far upstream region of the OVA promoter and CMV minimal promoter in combination with the Tet regulator.…”

Section: Inducible Expression Of Biologically Active Proteins In Tranmentioning

confidence: 99%

“…For instance, an intron within the vector construct may be removed. We experienced unexpected deletion caused by splicing of the vector sequence (Kodama et al 2012). In this case, the OVA promoter and its 5′ untranslated region ( Fig.…”

Section: Entry Entrymentioning

confidence: 99%

Transgenic chickens

Nishijima

Iijima

2012

Dev Growth Differ

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The development of transgenic chicken technology has lagged far behind that of mammalian species. Two reasons for this are that only a one-cell-stage oocyte can be obtained from a sacrificed hen and that the yolk prevents high-magnification microscopic observation of oocytes. Recently, several new methods have been developed that will enable the successful establishment of transgenic chickens. Retroviral vectors are used in many cases because of their ability to incorporate transgenes into host cell chromosomes in a highly efficient manner. These viral vectors are injected directly into the embryos, usually at the blastodermal stage. In some cases, primordial germ cells (PGCs) are infected in vitro and then implanted into recipient embryos. Methods that do not rely on retroviral vectors are also available for creating transgenic chickens. Long-term culture of PGCs permits the selection of stably transfected cells and implantation of the manipulated PGCs. In addition, embryonic stem (ES) cell systems are available; however, the induction of functional gametes from ES cells has not, to our knowledge, been successful. It is clear that recent developments suggest that chickens may be used as a valuable experimental genetic system.

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Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system

Cited by 18 publications

References 33 publications

Influence of Ovalbumin Gene Regulatory Elements on Tissue Specificity and Level of Transgene Expression

Influence of Ovalbumin Gene Regulatory Elements on Tissue Specificity and Level of Transgene Expression

Enhanced lentiviral vector production in 293FT cells expressing Siglec-9

Transgenic chickens

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