Enhanced Green Fluorescent Protein Gene Research Articles

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.

Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation

The amygdala is located in the caudal part of the ventral telencephalon. It is composed of many subdivisions and is involved in the control of emotion. It is important to know the mechanisms of amygdalar development in order to analyze the pathogenesis of emotional disorders, but they are still not adequately understood. In the present study the migration, differentiation, and distribution of amygdalar neurons in the mouse embryo were investigated by means of in utero electroporation. Ventricular zone cells in restricted regions, that is, the caudal ganglionic eminence (CGE), the ventral pallium, the lateral pallium, and the diencephalon, were labeled with an expression vector of the enhanced green fluorescent protein (EGFP) gene. Labeling at embryonic day (E)10 revealed that the central nucleus originates from the neuroepithelium in the ganglionic eminence and the labeling at E11 and E12 revealed that the basolateral complex originates from the neuroepithelium of the ventral and lateral pallia. The introduction of the EGFP gene into the neuroepithelium of the third ventricle at E11 showed that the medial nucleus originates, at least in part, from the neuroepithelium of the diencephalon and migrates over the diencephalo-telencephalic boundary. The radial glial arrangement corresponded well with the initial migration of amygdalar neurons, and the radial processes later formed the boundary demarcating the basolateral complex. These findings indicate that the neurons originating from the temporally and spatially restricted neuroepithelium in both the telencephalon and diencephalon migrate and differentiate to form the mosaic of amygdalar subdivisions.

312 CONSTRUCTION OF SPECIFICALLY EXPRESSED VECTOR IN MAMMARY GLAND FOR lacS AND ITS TRANSFECTION INTO BOVINE FETAL FIBROBLASTS MEDIATED BY LIPOSOME

This study was designed to optimize conditions for transfection of a mammary gland specific transgene into bovine fetal fibroblasts. Transfection of Sulfolobus solfataricus β-glycosidase gene (lacS) was mediated by liposome. Neomycin resistance (Neor) and enhanced green fluorescent protein (EGFP) gene were used as genetic markers to screen transgenic somatic cells. A 0.92-kb fragment of bovine β-lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and was inserted into the T site of pMD19-Simple T plasmid. 1.49 kb of lacS gene coding sequence was cloned from Sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor was derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid, thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovine β-lactoglobulin promoter for specific expression in mammary gland. Bovine fetal fibroblasts (bFF) were isolated from the ear skin of female fetuses at the age of 2 to 3 months. The cells proliferated well and grew normally in culture, with typical fibroblast morphology and growth curve. The effects of different concentrations of transfection and pBLI were compared on the efficiency of transfection. The passage 4 bFF cells at 70 to 80% confluency were transfected in a 24-well culture plate. 2 × 105 cells were cultured in DMEM with 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 μg of pBLI using transfection (1, 2, 3, 4, 5, and 6 μL) for 48 h, respectively. The transfected cells were cultured for 48 h before adding G418 at concentrations of 200, 300, 400, 500, 600, 700, 800, and 900 μg mL–1 for 14 d, respectively. Positive cell colonies were selected and purified through both the expression of Neor and EGFP gene under a fluorescence microscopy. The selected colonies were propagated in DMEM containing 300 μg mL–1 G418. The results showed that bright green fluorescence could be detected at 48 h after transfection. 1.0 μg of pBLI plasmid and 3 μL of transfection yielded the desirable efficiency of transfection. More transgenic bFF colonies were selected by G418 at the concentration of 800 μg mL–1. In conclusion, a specifically expressed vector in mammary gland for lacS gene was successfully constructed, transfection parameters were developed, and efficient screening measures were established for detecting transgenic somatic cells.

Stable EGFP gene expression in C6 glioma cell line after transduction with HIV-1-based lentiviral vector

To establish a stable C6/EGFP glioma cell line for studies on glioma. The C6 glioma cell line was transfected with the human immunodeficiency virus type I (HIV-1) based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein (EGFP)-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction (PCR) and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Transgenic breeding of anti-Bombyx moriL. nuclear polyhedrosis virus silkwormBombyx mori

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.

Transgenic breeding of anti-<italic>Bombyx mori</italic> L. nuclear polyhedrosis virus silkworm <italic>Bombyx mori</italic>

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.

Effects of lipid shell microbubble on ultrasound mediated EGFP gene delivery to transplanted tumors: initial experience

Objective To investigate the feasibility of ultrasound (US) mediated enhanced green fluorescent protein (EGFP) gene delivery in subcutaneous transplanted tumors of human cervical carcinoma (Hela) and the contribution of lipid shell microbubble (LSMB) on gene transfection.

Ex Vivo Magnetofection With Magnetic Nanoparticles: A Novel Platform for Nonviral Tissue Engineering

Several methods have been described to introduce DNA expression vectors into mammalian cells both in vitro and in vivo. Each system has benefits and limitations, and to date there is still no ideal method for gene transfer. In this study, we introduced a novel method of gene transfer by using Fe3O4 nanoparticles. The magnetic nanoparticles composed of Fe3O4, and the transfected genes used are Lac Z and enhanced green fluorescence protein gene (EGFG). Four different groups of preparations included in this study were homemade liposome-enveloped EGFP-DNA/Fe3O4, homemade liposome EGFP-DNA gene without magnetic Fe3O4 nanoparticles, lipofectamine 2000-enveloped EGFP-DNA, and EGFP-DNA gene only. Mice osteoblast and He99 lung cancer cell line were used as host cells for gene transfection. The time-dependent EGFP gene expression was monitored and analyzed. The results showed that the diameter of the complex was less than 100 nm. There was no cytotoxicity observed at any of the magnetic Fe3O4 nanoparticle concentrations tested. In the presence of magnetic field, the liposome-enveloped EGFP-DNA/Fe3O4 complex exhibited a much higher efficiency for transfecting EGFP-DNA into osteoblast cells under external magnetic fields. The gene can be transfected into cells with an aid of magnetic vectors and magnetic force. Under a gradient magnetic field, the efficiency of magnetofection is enhanced as compared to that without magnetic field.

Localization and targeting mechanisms of two chloroplastic β‐carbonic anhydrases in the marine diatom Phaeodactylum tricornutum

Two beta-CA genes, ptca1 and ptca2, were previously isolated from the marine diatom Phaeodactylum tricornutum and shown to be regulated by ambient [CO(2)] at the transcriptional levels. The product of ptca1, PtCA1, was also shown to be localized to the chloroplast as clumped particles. Both ptca1 and 2 encode putative N-terminal signal peptides of 19 amino acids. In the present study, the C-terminal region of the 46 amino acid presequence of PtCA1 (Pre46AA) was truncated to investigate the function of this region. DNA sequences, which encode the N-terminal 19, 23, 24, 34 or 44 polypeptides of Pre46AA, were fused with the sequence that encodes the mature protein, PtCA1. These constructs were fused with the enhanced-green fluorescent protein gene, egfp, and expressed in P. tricornutum. Fluorescent microscopy of expressed green fluorescent protein (GFP) showed that presequences of PtCA1 longer than the initial N-terminal 23 amino acids successfully sorted the PtCA1::GFP fusion into chloroplasts, whereas those shorter than 23 amino acids did not. These results indicate that the four amino acids after the endoplasmic reticulum signal peptide cleavage site are required as a chloroplast-targeting signal. Ala(19) Phe(20) is known to be the highly conserved transit motif among presequences of nuclear-encoded chloroplastic proteins in diatoms; however, the corresponding amino acid sequence deduced from the ptca2 was Ala(19) Leu(20). Substitution of Phe(20) in PtCA1 with Leu did not alter the localization of PtCA1, indicating that the chloroplast-targeting motif possesses some alternative pattern and that PtCA2 could be sorted to chloroplast via a process similar to that of PtCA1. The ptca2 cDNA was isolated, fused at the 3' terminus with the egfp and introduced into P. tricornutum cells. Expressed PtCA2::GFP fusion was clearly localized by chlorophyll fluorescence in the chloroplast of P. tricornutum as clumped particles, which was strikingly similar to the localization of PtCA1.

Localization of green fluorescent protein in mouse preimplantation embryos

The localization of enhanced green fluorescent protein (EGFP) was studied in preimplantation embryos obtained from reciprocal mating of hemizygous C57Bl/6-Tgn (ACTbEGFP)1Osb/J mice with C57Bl/6 mice. Specific fluorescence of EGFP was observed in all oocytes and embryos obtained from transgenic females during all preimplantation stages and in embryos inheriting the EGFP gene from transgenic males starting from the 8 blastomere stage during the compactization period. EGFP mRNA or EGFP synthesized during oogenesis can be retained in embryos during the entire preimplantation period, while expression of EGFP gene transferred from the father coincides with the onset of compactization. The possibility of using these embryos in experimental mammalian embryology is discussed.

In Vivo Gene Transfer of PPAR.GAMMA. Is Insulfficient to Induce Adipogenesis in Skeletal Muscle

Skeletal muscle contains several progenitor/stem cells with myogenicity as well as adipogenicity such as satellite cells. Our previous study demonstrated that forced expression of PPAR gamma is sufficient to induce transdifferentiation of predetermined myoblasts in vitro. In the present study, we examined whether introduction of PPAR gamma gene could induce adipogenesis of satellite cells in vivo. A plasmid vector containing enhanced green fluorescent protein (EGFP) or PPAR gamma gene was introduced into rat tibialis anterior muscle by electroporation. Histological analyses revealed that electroporation induces degenerative/regenerative response in skeletal muscle, including activation of satellite cells. When EGFP gene was introduced, newly formed myotubes resulted from fusion of activated satellite cells, showed EGFP expression, indicating that electroporation could transfect satellite cells with exogenously introduced gene. Gene transfer of PPAR gamma resulted in an increase of PPAR gamma-positive mononucleated cells on day 3 after electroporation but failed to induce adipogenesis thereafter. These results suggested that, in addition to an expression of PPAR gamma, niches that support adipogenesis are required for satellite cells to enter adipogenesis in vivo.

Functional analysis of the Myostatin gene promoter in sheep

Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNPro(W)-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNPro(E(3+5+7)M)-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3-1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin promoter.

Circadian Transcription Depends on Limiting Amounts of the Transcription Co-activator nejire/CBP

The circadian clock orchestrates physiological and behavioral activities, including metabolism, neuronal activity, and cell proliferation in synchrony with the environmental cycle of day and night. Here we show that the Drosophila ortholog of the CBP/p300 family of transcription co-activators, nejire (nej), is an intrinsic component of the circadian clock that performs regulatory functions for circadian controlled transcription. Screening of overexpression mutants revealed that gain of nej function was associated with a loss of behavioral and molecular rhythms. Overexpression of NEJ suppresses the long period phenotype of a mutation in the clock gene period (per). NEJ physically interacts through two binding sites with CLOCK and the CLOCK. CYCLE (CLK.CYC) complex. Induction of CLK.CYC-dependent transcripts upon induction of nej expression from a heat-shock promoter showed that NEJ is limiting. Reduced CLK.CYC-mediated transcription in a nej hypomorphic mutant indicates an essential function of NEJ/CBP for CLK.CYC activity and a regulation of circadian transcription by availability of the co-activator. Competition for recruitment of NEJ/CBP provides a potential mechanism for cross-talk between circadian transcription and other CBP-dependent physiological processes.

Tumor-specific Activation of Human Telomerase Reverses Transcriptase Promoter Activity by Activating Enhancer-binding Protein-2β in Human Lung Cancer Cells

The up-regulated expression and telomerase activity of human telomerase reverse transcriptase (hTERT) are hallmarks of tumorigenesis. The hTERT promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. However, little is known about how tumor cells differentially activate hTERT transcription and induce telomerase activity. In this study, we identified activating enhancer-binding protein-2beta (AP-2beta) as a novel transcription factor that specifically binds to and activates the hTERT promoter in human lung cancer cells. AP-2beta was detected in hTERT promoter DNA-protein complexes formed in nuclear extracts prepared only from lung cancer cells but not from normal cells. We verified the tumor-specific binding activity of AP-2beta for the hTERT promoter in vitro and in vivo and detected high expression levels of AP-2beta in lung cancer cells. We found that ectopic expression of AP-2beta reactivated hTERT promoter-driven reporter green fluorescent protein (GFP) gene and endogenous hTERT gene expression in normal cells, enhanced GFP gene expression in lung cancer cells, and prolonged the life span of primary lung bronchial epithelial cells. Furthermore, we found that inhibition of endogenous AP-2beta expression by AP-2beta gene-specific small interfering RNAs effectively attenuated hTERT promoter-driven GFP expression, suppressed telomerase activity, accelerated telomere shortening, and inhibited tumor cell growth by induction of apoptosis in lung cancer cells. Our results demonstrate the tumor-specific activation of the hTERT promoter by AP-2beta and imply the potential of AP-2beta as a novel tumor marker or a cancer therapeutic target.

A universal transgene silencing approach in baculovirus–insect cell system

Baculovirus–insect cell system (BICS) is considered one of the most efficient eukaryotic gene expression systems. This system has also been used for producing different recombinant baculoviruses with increased insect toxicity as potential biopesticides. Establishing a universal gene silencing (UGS) system is very important due to the increasing number of studies using RNA interference (RNAi) with BICS. In this work, the enhanced green fluorescent protein (EGFP) was used as the RNAi consistent target sequence located downstream of a depressant insect-neurotoxin gene, LqqIT2 used as a model of the gene of interest. Small interfering RNA (siRNA) and inverted repeats of EGFP gene (IR-EG) were examined in targeting the EGFP-LqqIT2 (EL)-fusion mRNA or LqqIT2-EGFP (LE)-bicistronic mRNA for degradation. Suppression efficiencies using these inducers were examined transiently and stably in uninfected and infected insect Sf9 cells. Moreover, RNAi showed persistence for 4 and 8 days in baculovirus-infected as well as uninfected Sf9 cells, respectively. Bicistronic RNA seems an efficient way to lower cost and effort of the gene silencing approach while maintaining the biological activity of the protein of interest when the RNAi is not in use.

Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA

We established a rapid procedure for obtaining transgenic mice by directly injecting an enhanced green fluorescent protein (EGFP)-expressing plasmid (pIRES-EGFP) into the ovaries of fertile mice. The frequency of transgenic mouse production was determined by pair-mating, and by polymerase chain reaction (PCR) and sequence analysis of DNA taken from the tails of the offspring. The mice that received the EGFP gene transmitted it to their offspring (F 1). Genetic and PCR analyses of F 1 progeny confirmed that the inserted EGFP was stably inherited. Of six female F 1 mice, all were able to pass the foreign DNA on to the next generation (F 2). In situ hybridization using paraffin-embedded sections of ovarian and testicular tissues from the F 1 and F 2 progeny showed that the introduced gene was expressed in the gonads of the animals. The chromosomal location of the injected DNA was determined by fluorescence in situ hybridization, and the frequency of multiple site versus single site insertions is 85.71% (18/21) analyzed by FISH. We anticipate great progress in murine genetic engineering using this technique.

In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.

PROTOPLAST CO-TRANSFORMATION AND REGENERATION OF TRANSGENIC 'HAMLIN' SWEET ORANGE PLANTS CONTAINING A CDNA XA21 XANTHOMONAS RESISTANCE GENE AND GFP

[...] Plasmid DNA (pARS108) encoding the non-destructive selectable marker EGFP (Enhanced Green Fluorescent Protein) gene, and the plasmid cDNA of the Xa21 gene (pXa21-mtaq) were co-transformed into 'Hamlin' orange protoplasts using polyethylene glycol. More than 150 transgenic embryoids were recovered. Over a thousand transgenic plantlets GFP positive were regenerated from 150 independent transformation events. PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in some of the transgenic plantlets. The recovery of transgenic plants was expedited by in vitro grafting.[...]

Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in A Promoter-Dependent Manner

Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters--cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken beta-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1alpha (EF1alpha)--in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.