Improved expression of green fluorescent protein in cattle embryos produced by ICSI-mediated gene transfer with spermatozoa treated with streptolysin-O

The presence of Alu repeats downregulates the expression of the green fluorescent protein (GFP) gene. We found that SV40PolyA (PolyA, 240 bp), in either orientation, eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats. In this study, 4 different segments (each 60 bp) were amplified from antisense PolyA (PolyAas) by PCR, and inserted upstream of Alu14 in pAlu14 plasmid (14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in a head-tail tandem manner). Segments 1F1R (the first 60 bp segment at the 5' end of PolyAas) and 4F4R (the fourth 60 bp segment from the 5' end of PolyAas) did not activate GFP gene expression, whereas 2F2R and 3F3R (the middle two segments) did (as detected by Northern blot analysis and fluorescent microscopy). Different copy numbers of 2F2R and 3F3R segments, in a head and tail tandem manner, were inserted downstream of the GFP gene in pAlu14. p2F2R*4-Alu28, p3F3R*4-Alu18 and p3F3R*4-Alu28 were used as length controls to verify that the decrease in the expression of GFP was not due to the increased length of the inserted segment in the expression vectors. We found that 2 and 4 copies of 2F2R or 3F3R activated the GFP gene more strongly than one copy of them. However, more than 8 copies of 2F2R or 3F3R reduced the activation of the GFP gene. We concluded that SV40PolyAas contained at least two gene-activating elements (2F2R and 3F3R) and 2-4 copies of 2F2R or 3F3R were optimal for the expression of the GFP gene.

Effects of additional sequences directly downstream from the AUG on the expression of GFP gene

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

It is necessary to use a suitable marker gene to select embryos expressing transgene prior to transfer into recipient females especially in an efficient production of transgenic farm animals. Recently a green fluorescent protein (GFP) gene has been used as a marker gene for this purpose, because it is a self-fluorescent substance and does not require a substrate such as X-Gal in a lacZ gene. Using these characteristics of GFP, we transfected the GFP gene with the neomycin-resistance gene under the control of the phosphoglycerase kinase promoter into mouse embryonic stem (ES) cells, and attempted to produce a transgenic mouse derived from aggregates of GFP-positive ES cells after the selection with G418 for 8 days with diploid or tetraploid embryos. There were some ES colonies expressing GFP at different levels after the selection with G418. These GFP-positive ES cells were used as a complete tight adhered clump for aggregation with diploid or tetraploid embryos and resulted in GFP expression in the inner cell mass (ICM) cells of aggregates, but no embryos expressing GFP in trophoblasts. At 9.5 days p.c., 6 out of 10 live fetuses (60%) for diploid⇔ES chimera, and all of 14 live fetuses (100%) for tetraploid⇔ES chimera, in spite of head deformity, were transgenic fetuses. At 14.5 days p.c., among 7 normal embryos obtained from 21 embryos transferred to recipients, one fetus showed GFP expression in five organs (heart, kidney, liver, gonad and intestine), and 4 fetuses strongly expressed GFP in more than one organ as seen under the fluorescent dissecting microscope. These results indicate that enough transfected ES cells selected in a short time with G418 were able to be used for the selection of transgenic embryos prior to transfer to the recipient. Moreover, by using a complete tight adhered clump of ES cells for aggregation, the clump is able to completely contribute to a part of the ICM. The GFP gene may be useful and efficient as a marker especially for farm animals which have a long term pregnancy and a small litter size.

Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Umbilical cord blood (CB) from the early gestational human fetus is recognized as a rich source of hematopoietic stem cells. To examine the value of fetal CB for gene therapy of inborn immunohematopoietic disorders, we tested the feasibility of genetic modification of CD34(+) cells from CB at weeks 24 to 34 of pregnancy, using lentiviral vector-mediated transfer of the green fluorescent protein (GFP) gene. The transduction rate of CD34(+) cells was 42 +/- 9%, resulting in GFP expression in 23 +/- 4% of colonies derived from colony-forming units (CFUs) and 11 +/- 1% from primitive long-term culture-initiating cells (LTC-ICs). Cell cycle analysis demonstrated transduction and GFP expression in cells in the G(0) phase, which contains immature hematopoietic progenitors. Transduced fetal CD34(+) cells could be expanded 1000-fold in long-term cultures supplemented with megakaryocyte growth and development factor along with Flt-3 ligand. At week 10, expression of GFP was observed in 40.5 +/- 11.7% of CFU-derived colonies. While prestimulation of CD34(+) cells with cytokines prior to transduction increased the efficiency of GFP transfer 2- to 3-fold, long-term maintenance of GFP-expressing CFUs occurred only in the absence of prestimulation. The GFP gene was found integrated into the genomic DNA of 35% of LTC-IC-derived colonies initiated at week 10, but GFP expression was not detectable, suggesting downregulation of transgene activity during the extended culture period. These results indicate that human fetal CB progenitors are amenable to genetic modification by lentiviral vectors and may serve as a target for gene therapy of hematopoietic disorders by prenatal autologous transplantation.

One of the critical problems to be solved in transgenic animal production is non-controllable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP (green fluorescent protein) gene under the control of tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence was also introduced into the retrovirus vector downstream of either the GFP gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator). Transformed cells derived from porcine fetus were cultured in the medium supplemented with or without doxycycline (tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at the 3′ side of the GFP gene, while tighter expression control (up to 20-fold) was obtained from the vector in which the WPRE sequence was placed at the 3′ side of the rtTA sequence. Encouraged with these data, we substituted the hPTH (human parathyroid hormone) gene for the GFP gene in the retrovirus vector. The porcine fetal fibroblast cells transformed by the modified retrovirus vector secreted hPTH into the medium under the tight control of doxycycline as observed in GFP expression. The resulting porcine cells secreting hPTH will be used in nuclear transfer experiment. This study was financially supported by the National Livestock Research Institute RDA (Suwon 441-350, Korea), ARPC (Agriculture R & D Promotion center, 2002–2005), and by grant No. R11-2002-100-01000-0 from the ERC program of the Korea Science & Engineering Foundation.

Robust In Vivo Transduction of Nervous System and Neural Stem Cells by Early Gestational Intra Amniotic Gene Transfer Using Lentiviral Vector

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierce's disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.

Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell-parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays. GFP, beta-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope. Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein. Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays.

The green fluorescent protein (GFP) gene was administered to intraperitoneally (i.p.) growing human stomach cancer in nude mice to visualize future regional and distant metastases. GFP retroviral supernatants were injected i.p. from day 4 to day 10 after i.p. implantation of the cancer cells. Tumor and metastasis fluorescence was visualized every other week with the use of fluorescence optics via a laparotomy on the tumor-bearing animals. At 2 weeks after retroviral GFP delivery, GFP-expressing tumor cells were observed in gonadal fat, greater omentum, and intestine, indicating that these primary i.p. growing tumors were efficiently transduced by the GFP gene and could be visualized by its expression. At the second and third laparotomies, GFP-expressing tumor cells were observed to have spread to lymph nodes in the mesentery and other regional sites. At the fourth laparotomy, widespread tumor growth was visualized by GFP expression, inducing liver metastasis. No normal tissues were found to be transduced by the GFP retrovirus. Thus, reporter gene transduction of the primary tumor enabled detection of its subsequent metastasis. This gene therapy model could be applied to primary tumors before resection or other treatment to have a fluorescent early detection system for metastasis and recurrence.

The green fluorescent protein (GFP) gene was administered to intraperitoneally (i.p.) growing human stomach cancer in nude mice to visualize future regional and distant metastases. GFP retroviral supernatants were injected i.p. from day 4 to day 10 after i.p. implantation of the cancer cells. Tumor and metastasis fluorescence was visualized every other week with the use of fluorescence optics via a laparotomy on the tumor-bearing animals. At 2 weeks after retroviral GFP delivery, GFP-expressing tumor cells were observed in gonadal fat, greater omentum, and intestine, indicating that these primary i.p. growing tumors were efficiently transduced by the GFP gene and could be visualized by its expression. At the second and third laparotomies, GFP-expressing tumor cells were observed to have spread to lymph nodes in the mesentery and other regional sites. At the fourth laparotomy, widespread tumor growth was visualized by GFP expression, inducing liver metastasis. No normal tissues were found to be transduced by the GFP retrovirus. Thus, reporter gene transduction of the primary tumor enabled detection of its subsequent metastasis. This gene therapy model could be applied to primary tumors before resection or other treatment to have a fluorescent early detection system for metastasis and recurrence.

Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection