Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts

Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.     

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Two human MARs effectively increase transgene expression in transfected CHO cells

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR‐3 and MAR‐7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real‐time PCR. The results showed that the expression level of eGFP of cells transfected with MAR‐containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR‐7 was higher than that of MAR‐3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR‐3 and MAR‐7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.     

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.     

A one-plasmid conditional color-switching transgenic system for multimodal bioimaging

We have developed a new construct to generate transgenic mice with one plasmid that offers: (1) Cre/loxP-mediated spatial and temporally-controlled tissue-specific transgene expression; (2) A color-switching mechanism that uses spectrum-complementary genetically-encoded red (mRFP) and green (eGFP) fluorescent markers to label the transgene-expressing cells; (3) A bioluminescent marker that turns-on in the transgene-expressing cells; (4) eGFP as a cell surface marker in the transgene-expressing cells that facilitates the isolation and targeting of these cells. This vector was tested in vitro by co-transfection of the transgenic plasmid and a plasmid containing Cre recombinase into cultured cells and by establishing a transgenic mouse line. We show that this method allows versatile transgene expression targeting and color-switching to facilitate fluorescent and bioluminescent imaging both in cultured cells and in vivo. Our strategy provides time-saving features in tissue-specific transgene expression, bioimaging and primary cell isolation and can be used for generation of gene-specific transgenic mice.     

A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells

A chimeric DNA fragment containing an interferon-beta matrix attachment region (MAR) and an immunoglobulin MAR (PSAR2) was synthesized. PSAR2 was cloned into the upstream or downstream region of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, which was then transfected into CHO cells. The results showed that PSAR2 did not effectively increase transgene expression when it was cloned into the upstream region of the eGFP expression cassette. However, when inserted downstream of the eGFP expression cassette, PSAR2-enhanced transient transgene expression and significantly increased the numbers of stably transfected cells compared with the control vector. Additionally, PSAR2 significantly increased eGFP copy numbers as compared with the control vector. PSAR2 could significantly enhance transgene expression in CHO cells according to the position in the vector and increased transgene copy numbers. We found a short chimeric sequence harboring two MARs effectively increased transgene expression in CHO cells.     

Production of Pigs Expressing a Transgene under the Control of a Tetracycline-Inducible System

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.     

Generation of transgenic dogs that conditionally express green fluorescent protein

We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.     

SV40 intron,a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF‐1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT‐PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.     

利用ES细胞建立可诱导的白血病模型

胚胎干细胞（embryonic stem cells,ESCs）是从囊胚的内细胞团分离出来的多潜能干细胞,具有多向分化的能力。将外源基因导入ES细胞建立转基因动物,对于研究外源基因的功能和调控具有一定的价值。载有外源性基因的病毒在感染ES细胞后,可通过囊胚注射获得具有胚系遗传的该转基因动物,并且这一外源基因可以稳定遗传和表达。该研究主要是利用携带hPML-RARα基因的慢病毒感染小鼠ES细胞系（R1）,获得携带该基因的ES细胞,感染后的ES细胞核型正常。在此基础上,将感染后的ES细胞经囊胚注射,获得了携带有hPML-RARα基因的3只嵌合小鼠,其中,有1只具有遗传特性。对嵌合体小鼠与C57杂交的后代给予强力霉素（doxycycline）处理,3天以后骨髓细胞hPML-RARα基因开始表达,这证明了在小鼠体内该外源基因表达的可诱导性。以上证实,已经成功利用ES细胞建立了可诱导的白血病转基因小鼠模型。     

Apoptosis of liver-derived cells induced by parvovirus B19 nonstructural protein

Parvovirus B19 has been implicated in some cases of acute fulminant non-A, non-B, non-C, non-G liver failure. Our laboratory previously demonstrated that B19 infection of hepatocytes induces apoptosis and that the B19 viral nonstructural protein, NS1, may play a critical role. To study the involvement of NS1 in apoptosis of liver cells, we generated a fusion protein of NS1 with enhanced green fluorescent protein (eGFP) in a system allowing for inducible gene expression. Transfection of the liver-derived cell line HepG2 with the eGFP/NS1 vector allowed expression of the fusion protein, which was visualized by fluorescence microscopy and demonstrated by immunoblotting. The fusion protein localized to discrete domains in the nucleus. Transfection of HepG2 cells with the eGFP/NS1 vector led to apoptosis of 35% of transfected cells, a sevenfold increase over cells transfected with the parent eGFP expression vector. Mutation of the eGFP/NS1 vector to eliminate the nucleoside triphosphate-binding site of NS1 significantly decreased apoptosis, as did treatment of transfected cells with inhibitors of caspase 3 or 9. Neutralization of tumor necrosis factor alpha or Fas ligand had no effect on apoptosis. These results demonstrate that NS1 is sufficient to induce apoptosis in liver-derived cells and that it does so through the initiation of an intrinsic caspase pathway.     

Efficient method for expressing transgenes in nonhuman primate embryos using a stable episomal vector

Transgenesis in the nonhuman primate can enhance the study of human biology by providing animal models for the study of primate-specific physiology, pathophysiology, and embryonic development. Progress with this technology has been hindered by the inherent inefficiency of transgenesis, transgene silencing, and practical restrictions on the production of sufficient pronuclear stage nonhuman primate zygotes. We have developed a novel technique using an Epstein Barr virus (EBV)-based episomal vector to produce rhesus monkey (Macaca mulatta) embryos expressing a transgene. Plasmid DNA containing the latent origin of replication, oriP, and Epstein Barr Nuclear Antigen-1 (EBNA-1) of EBV, as well as a CMV IE-enhanced green fluorescent protein (eGFP) expression cassette, was introduced into rhesus embryos by direct pronuclear microinjection. We detected eGFP in early cleavage stage embryos (4-8 cell) and throughout the duration of culture (day 8-9 blastocysts) by epifluorescent microscopy. A 50% transduction rate was obtained with the EBV-based vector. Microinjected embryos expressed eGFP and retained their developmental capacity as evidenced by development to the blastocyst stage. EBV-based vectors present a novel and efficient means of delivering transgenes for the study of the molecular control of primate embryonic development.     

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening.     

Conditional transgene expression mediated by the mouse beta-actin locus

Transgenic mice are an effective model to study gene function in vivo; however, position effects can complicate tissue-specific transgene analysis. To facilitate precise targeting of a transgenic construct into the mouse genome, we combined the Cre/lox and Flp/FRT recombination systems to allow for rapid transgene replacement and conditional transgene expression from the endogenous beta-actin locus. Flp/FRT recombination was used to rapidly exchange FRT-flanked transgene cassettes by recombinase-mediated cassette exchange in embryonic stem cells, while transgene expression can be activated in mice after Cre-mediated excision of a floxed STOP cassette. To validate our system, we analyzed the expression profile of an EGFP reporter gene after integration into the beta-actin locus and Cre-mediated excision of the floxed STOP cassette. Breeding of EGFP reporter mice with various Cre mouse lines resulted in the expected expression profiles, demonstrating the feasibility of the model to facilitate predictable and strong transgene expression in a spatially and temporally controlled manner.     

Production,purification and titration of a lentivirus-based vector for gene delivery purposes

Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at −80 °C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 × 10⁸ transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 10⁵ TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9652-5) contains supplementary material, which is available to authorized users.     

Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering

The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.