Highly efficient site-specific transgenesis in cancer cell lines.

BACKGROUND: Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. METHODS: According to this system, a "docking-site" was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an "incoming" vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. RESULTS: Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format. CONCLUSIONS: The novel PhiC31-IMSI system described in this study represents a powerful tool that can facilitate the characterization of cancer-related genes.

An isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by an unusual deletion with neomorphic activity. This deletion derepresses genes in cis; however which candidate gene causes the FSHD phenotype, and through what mechanism, is unknown. We describe a novel genetic tool, inducible cassette exchange, enabling rapid generation of isogenetically modified cells with conditional and variable transgene expression. We compare the effects of expressing variable levels of each FSHD candidate gene on myoblasts. This screen identified only one gene with overt toxicity: DUX4 (double homeobox, chromosome 4), a protein with two homeodomains, each similar in sequence to Pax3 and Pax7. DUX4 expression recapitulates key features of the FSHD molecular phenotype, including repression of MyoD and its target genes, diminished myogenic differentiation, repression of glutathione redox pathway components, and sensitivity to oxidative stress. We further demonstrate competition between DUX4 and Pax3/Pax7: when either Pax3 or Pax7 is expressed at high levels, DUX4 is no longer toxic. We propose a hypothesis for FSHD in which DUX4 expression interferes with Pax7 in satellite cells, and inappropriately regulates Pax targets, including myogenic regulatory factors, during regeneration.

Adjusting transgene expression levels in lymphocytes with a set of inducible promoters.

BACKGROUND: Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells. METHODS: The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells. RESULTS: The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels. CONCLUSIONS: The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications.

Management of upper abdominal laxity after massive weight loss: reverse abdominoplasty and inframammary fold reconstruction.

BACKGROUND: Central to body contouring after weight loss surgery is treatment of the abdominal region, often through a circumferential abdominoplasty. This procedure, however, neglects the laxity of the lower thoracic/upper abdominal region. A reverse abdominoplasty with reconstruction of a new inframammary fold (IMF) corrects this deformity through removal of excess skin along the IMF. Since 2002, we have performed 88 reverse abdominoplasty procedures within the context of a single or staged total-body lift (TBL). METHODS: A retrospective chart review of 129 TBL cases indicated that 88 patients had a combined or staged reverse abdominoplasty and circumferential abdominoplasty. Complication rates were noted as localized or generalized. RESULTS: Fifty-three of our patients had combined reverse abdominoplasty and circumferential abdominoplasty and 35 had the reverse abdominoplasty during a second stage. The complication rates for both groups were about 5% per patient per procedure with differences that were not statistically significant. Also, the revision rates for reverse abdominoplasty and circumferential abdominoplasty were similar for both groups, indicating patient satisfaction with the procedures. CONCLUSION: In selected patients, effective treatment of the abdominal region demands correction of both the upper and lower abdominal laxity and contour. This can be performed safely, effectively, and reliably by a reverse abdominoplasty with IMF reconstruction independently or simultaneously with circumferential abdominoplasty.

Enhanced recovery after body-contouring surgery: reducing surgical complication rates by optimizing nutrition.

BACKGROUND: Currently, many body-contouring patients are overweight or obese or recently have sustained massive weight loss. Often these patients need multiple surgical procedures with extensive incisions. The need for optimal healing in all these patient populations is, however, hampered by their existing nutritional deficiencies. METHODS: Based on the authors' previous work (Agha-Mohammadi and Hurwitz Plast Reconstr Surg 122:604-618, 2008; Agha-Mohammadi and Hurwitz Plast Reconstr Surg 122:1901-1914, 2008), three clinical studies were initiated. The first study examined the preoperative nutritional parameters of 90 body-contouring patients. Of the 48 postbariatric patients, 38% had low prealbumin (<20 mg/dl), 33% had vitamin A deficiency, 32.6% had low hemoglobin (<12 g/dl), 16.3% had iron deficiency, 9.5% had vitamin B12 deficiency, and 12% had hyperhomocystinemia. Among the 42 nonbariatric patients, only 10% had low prealbumin and 11.5% had vitamin A deficiency. Other deficiencies were less remarkable. The second study evaluated the effect of a surgical nutritional supplement on the prealbumin, vitamin A, vitamin B12, and folate levels of 13 decubitus ulcer patients. The data indicated an increase in all the measured parameters, particularly a prealbumin increase of about 0.8 mg/dl per day. The third study investigated complications experienced by 131 body-contouring patients. The patients were divided into postbariatric and nonbariatric groups, then stratified according to their body mass index (BMI). The rates of minor and major complications in obese nonbariatric and postbariatric patients (receiving the nutritional supplement) were comparable with those of normal-weight nonbariatric patients. CONCLUSION: Obese nonbariatric and postbariatric patients are at nutritional risk for many primary ingredients of wound healing and immune system competency. Appropriate nutritional supplementation is an effective means for correcting these nutritional parameters and can significantly reduce surgical complications associated with obesity and bariatric surgery.

A clinical review of total body lift surgery.

BACKGROUND: Until 2001, body contouring surgery after massive weight loss was uncoordinated, with few patients achieving compete rehabilitation. OBJECTIVE: The authors report a 5-year, retrospective, 75-case clinical review to determine the effectiveness, reliability, and safety of single and multistage total body lift (TBL) surgery. METHODS: Between January 2001 and June 2006, 59 single-stage, 15 two-stage, and 1 three-stage TBL surgeries were performed, involving a total of 605 separate procedures. Outcomes and complications were compared among all TBL patients and a contemporaneous published series. RESULTS: Three representative cases are described. Overall, in patients under 55 years of age with a body mass index of less than 30, there was no significant difference in the choice of procedure (ie, single-stage TBL [95% confidence interval, 1.236-2.302] or multiple-stage TBL [95% confidence interval, 1.687-4.892]; P = .1882). Although there was no significant association between major complications and the number of procedures performed in this cohort of patients, there were increased wound healing problems following multiple-stage TBL (P > .5). Single-stage TBL surgery averaged 8.4 hours. Two-stage surgery took 7.4 hours for the first stage and 4.6 hours for the second stage, for a total of 11 hours. Banked blood transfusions for single-stage surgery were 1.5 per single-stage case and 0.78 per multi-stage case. Seventy-six percent of the patients experienced complications, mostly related to wound healing. All preoperative and postoperative Pittsburgh rating grades improved. CONCLUSIONS: TBL is customized for individuals who desire a comprehensive approach to improvement of their loose skin. The rate of complications was high and comparable to other published series. There was no difference between the complications of the single-and two-stage patients. While there was an observable reduction in deformity and a high rate of satisfactory aesthetic outcomes, this high number of complications indicates a need to improve clinical performance.

A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines.

BACKGROUND: The urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines. RESULTS: A DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension. CONCLUSION: This doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.

Mesenchymal stem cells: aesthetic applications.

Adult stem cells may have significant aesthetic surgery applications. Their replicative capacity and plasticity may be useful in engineering autologous grafts for soft tissue and facial skeletal augmentation. Another possibility is that increasing the concentration of mesenchymal stem cells in the facial soft tissue at regular intervals during adulthood will maintain volume and elasticity. (Aesthetic Surg J 2003;23:504-506.).

Induced pluripotent stem cell lines derived from equine fibroblasts.

The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species.

Potential impacts of nutritional deficiency of postbariatric patients on body contouring surgery.

BACKGROUND: Bariatric surgery is currently the most effective method of sustainable weight loss for the morbidly obese patient. In the months to years that follow, many patients develop nutritional deficiencies of proteins, vitamins, and minerals as they present for plastic surgery and body contouring operations. METHODS: The aim of this review is to highlight the nutritional deficiencies of postbariatric patients as related to their planned body contouring surgery. This review was prepared by an extensive search of the PubMed and Ovid databases for terms such as "bariatric surgery," "nutritional deficiency," "wound healing," and "immune response." RESULTS: The current review indicates that many of the deficient macronutrients and micronutrients of postbariatric patients are implicated in wound healing and optimal immune response. CONCLUSIONS: Although the optimal nutrient intake to promote wound healing is unknown, it is clear that proteins, vitamin A, vitamin C, vitamin B complex, arginine, glutamine, iron, zinc, and selenium can have significant beneficial effects on wound healing and optimization of the immune system. Furthermore, vitamin B complex can reduce homocysteine levels, which is inversely associated with the risk of venous thrombosis and endothelial cell dysfunction. The authors' preliminary experience with a nutritional blend that contains all of the aforementioned ingredients has significantly reduced their complication rates.

Nutritional deficiency of post-bariatric surgery body contouring patients: what every plastic surgeon should know.

BACKGROUND: Bariatric surgery, particularly the Roux-en-Y gastric bypass, is currently the most effective method of sustainable weight loss for the morbidly obese patient population. Unfortunately, the nutritional adequacy of the postoperative diet has frequently been overlooked, and in the months to years that follow, many nutritional deficiencies have become apparent. Furthermore, once weight loss has reached a plateau, many patients become candidates for body contouring surgery and other aesthetic operations. METHODS: The aim of this review was to highlight the nutritional deficiencies of post-bariatric surgery patients as related to planned body contouring surgery. This review was prepared by an extensive search of the bariatric surgery literature. RESULTS: The current data indicate that many post-bariatric surgery patients have protein-calorie malnutrition as well as various vitamins and mineral deficiencies that may limit optimal health and healing. CONCLUSIONS: It is essential that those plastic surgeons who treat post-bariatric surgery patients are aware of these nutritional deficiencies, which can be minimized by adhering to eating guidelines and supplemental prescriptions. Although there are many studies documenting relationships between malnutrition and poor wound healing, the optimal nutrient intake in the post-bariatric surgery state to promote wound healing is unknown. It is, however, clear that proteins, vitamin A, vitamin C, arginine, glutamine, zinc, and selenium have significant beneficial effects on wound healing and optimizing the immune system.

Postbariatric surgery breast reshaping: the spiral flap.

INTRODUCTION: After massive weight loss, the breasts have poor shape, projection, and skin elasticity. Breast reshaping is recognized as difficult and may require excess nearby tissues. As the senior author's approach evolved over the past 4 years, breast reshaping with the spiral flap became integral to an upper body lift. MATERIALS AND METHODS: After the weight loss has stabilized, body contouring surgery has been performed on 53 patients over a 3-year period. Six patients had mastopexy and/or augmentation only. Eighteen patients had spiral flap breast reshaping as part of an upper body lift. This lift is a reverse abdominoplasty that ends along the inframammary fold incision of the Wise pattern mastopexy and continues laterally to along the back roll. Excess tissue from the epigastrium and lateral back roll is deepithelialized and used for augmentation. These flap extensions of the central breast pedicle are spiraled around the breast for augmentation, shaping, and suspension. When more tissue is needed, saline-filled silicone implants have been used, preferably during a second stage. RESULTS: Follow up of this initial group ranged from 4 to 28 months with a mean of 11 months. In this initial effort, 14 of the 18 were pleased. In 3 patients, subsequent bilateral saline implants further augmented the breasts. Tip fat necrosis was evident by firmness of the tissues in 3 patients and resolved in all but 1. That 1 patient had operative debridement of the distal 50% of the flaps followed by saline-filled silicone implants. One patient was disappointed with the back scar. Two patients dislike the shape and fill of their breast and have not returned for revision. CONCLUSION: During 3 years of focused clinical activity, we have evolved the spiral flap reshaping with upper body lift into a comprehensive, effective, satisfying, and safe esthetic contouring of the breast and upper torso after massive weight loss performed with an upper body lift.

Breast reconstruction with alloplastic implants.

This article reviews immediate and delayed breast reconstruction with prosthetic implants, and the effect of irradiation therapy. Despite widespread use of breast conserving surgery for early breast cancer, many breast cancer patients still undergo mastectomy. Some of these patients choose breast reconstruction. Over the last 30 years, techniques for breast reconstruction have evolved significantly with new alternative techniques and improved surgical devises. Immediate or delayed breast reconstruction with silicone prosthesis can be an excellent option. Implant reconstruction may be single or two stage procedures. Traditionally, small breasts with minimal ptosis are suited for single-stage reconstruction. Large breasts or inadequate skin require expanders followed by implants. Minimal excision mastectomy and biological spacers are allowing larger breast single stage reconstruction and improved aesthetics for two stage procedures. With recent studies suggesting survival advantage of post-mastectomy irradiation, many candidates for breast reconstruction are receiving radiotherapy, which complicates healing after breast reconstruction.

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters.

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.

Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness.

BACKGROUND: The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency. METHODS: To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A. RESULTS: In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (P(hCMV*-1)) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original P(hCMV*-1) in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses.