Transduction of the mammary epithelium with adenovirus vectors in vivo

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.     

Transgene delivery via intracellular electroporetic nanoinjection

Development of an effective cytoplasmic delivery technique has remained an elusive goal for decades despite the success of pronuclear microinjection. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In this work we report a cytoplasmic delivery method termed intracellular electroporetic nanoinjection (IEN). IEN is unique in that it manipulates transgenes using electrical forces. The microelectromechanical system (MEMS) uses electrostatic charge to physically pick up transgenes and place them in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronuclei using electrical pulses. Standard electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm for transgene delivery from the cytoplasm to the pronucleus. In this report we describe the principles which allow localized electroporation of the pronuclei including: the location of mouse pronuclei between 21 and 28 h post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on electric-field-driven movement of transgenes. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.     

Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism. J. Cell. Physiol. 174:251–260, 1998. © 1998 Wiley-Liss, Inc.     

High-level expression of human lactoferrin in the milk of goats by using replication-defective adenoviral vectors

The expression of human lactoferrin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce lactorferrin in the milk of transgenic animals resulted in very high cost and uncertain results. In this paper, we have directly infused replication-defective adenovirus encoding human lactoferrin cDNA into the mammary gland of goats via the teat canal. In this way, we obtained a high level of expressed human lactoferrin up to 2g/L in the milk of goats. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. A approximately 80-kDa protein was visualized after viral vector infection. Our results demonstrate that intraductal injection of recombinant replication-defective adenovirus vectors may provide a very useful tool for large-scale production of recombinant proteins of biopharmaceutical interest.     

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-κB) signaling, as visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse¹.Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration^2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-κB luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-κB signaling within the LPS-injected gland⁴.These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.     

乳腺注射法表达人组织型纤溶酶原激活剂的研究

目的 :构建tPA乳腺定位表达载体 ,使其在牛乳汁中高效表达 ,观察目的基因表达的规律及其影响因素 ,为建立新型牛乳腺生物反应器提供理论基础。方法 :RT-PCR法克隆目的基因 ,通过酶切、连接、分离、纯化等方法构建含tPA-cDNA的乳腺定位表达载体 ;采用乳腺注射法将融合基因转入小鼠及牛的乳腺组织中。结果 :乳腺注射外源基因后 ,tPA可在小鼠和牛的乳汁中表达。结论 :乳腺注射法可使目的基因在乳腺组织中稳定地表达较长的时间 ,其表达量与显微注射法没有明显的差异 ,表明外源基因的表达不受转基因方法的影响。但tPA在牛乳汁中的表达量明显高于小鼠的表达量 ,提示不同动物的乳蛋白调控系统有一定的差异 ,可能受着不同的因素或调控系统的影响。     

Transient expression of hepatitis B virus surface antigen (HBsAg) and human erythropoietin (hEPO) genes in milk after direct introduction into ewe

Ｅｘｐｒｅｓｓｉｏｎｏｆｍｉｌｋｐｒｏｔｅｉｎｇｅｎｅｓｉｓｉｎｖｏｌｖｅｄｉｎａｈｕｇｅｎｅｔｗｏｒｋｏｆｒｅｇｕｌａｔｏｒｙｃｉｒｃｕｉｔｓｗｈｉｃｈａｒｅｌｉｎｋｅｄｔｏｔｈｅｉｎｔａｃｔｄｅｖｅｌｏｐｉｎｇｍａｍｍａｒｙｇｌａｎｄ,ａｎｄｈｏｍｅｏｓｔａｓｉｓｄｕｒｉｎｇｐｕｂｅｒｔｙ,ｐｒｅｇｎａｎｃｙ,ｌａｃｔａｔｉｏｎａｎｄｉｎｖｏｌｕｔｉｏｎ.Ａｎａｌｙｓｉｓｏｆｐｕｔａｔｉｖｅｒｅｇｕｌａｔｏｒｙｅｌｅｍｅｎｔｓａｎｄｈｙｂｒｉｄｇｅｎｅｉｎｔｉｓｓｕｅｃｕｌｔｕｒｅｓｙｓｔｅｍｓ…     

Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. The procedure involves rapid, high-pressure injection of a DNA solution into the tail vein. The overall procedure takes <1 h although the delivery into one mouse requires only a few seconds. Successful injections result in expression of the transgene in 5-40% of hepatocytes 1 d after injection. Several weeks after injection, transgene expression stabilizes at approximately 1% of the level at 24 h, presumably owing to integration of the transposons into chromosomes.     

Permeabilization of the Blood-Brain Barrier via Mucosal Engrafting: Implications for Drug Delivery to the Brain

Utilization of neuropharmaceuticals for central nervous system(CNS) disease is highly limited due to the blood-brain barrier(BBB) which restricts molecules larger than 500Da from reaching the CNS. The development of a reliable method to bypass the BBB would represent an enormous advance in neuropharmacology enabling the use of many potential disease modifying therapies. Previous attempts such as transcranial catheter implantation have proven to be temporary and associated with multiple complications. Here we describe a novel method of creating a semipermeable window in the BBB using purely autologous tissues to allow for high molecular weight(HMW) drug delivery to the CNS. This approach is inspired by recent advances in human endoscopic transnasal skull base surgical techniques and involves engrafting semipermeable nasal mucosa within a surgical defect in the BBB. The mucosal graft thereby creates a permanent transmucosal conduit for drugs to access the CNS. The main objective of this study was to develop a murine model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain. Using this model we demonstrate that mucosal grafts allow for the transport of molecules up to 500 kDa directly to the brain in both a time and molecular weight dependent fashion. Markers up to 40 kDa were found within the striatum suggesting a potential role for this technique in the treatment of Parkinson’s disease. This proof of principle study demonstrates that mucosal engrafting represents the first permanent and stable method of bypassing the BBB thereby providing a pathway for HMW therapeutics directly into the CNS.     

Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal mice

BACKGROUND: Direct injection of lentivectors into the central nervous system (CNS) mostly results in localized parenchymal transgene expression. Intrathecal gene delivery into the spinal canal may produce a wider dissemination of the transgene and allow diffusion of secreted transgenic proteins throughout the cerebrospinal fluid (CSF). Herein, we analyze the distribution and expression of LacZ and SEAP transgenes following the intrathecal delivery of lentivectors into the spinal canal. METHODS: Four weeks after intrathecal injection into the spinal canal of newborn mice, the expression of the LacZ gene was assessed by histochemical staining and by in situ polymer chain reaction (PCR). Following the spinal infusion of a lentivector carrying the SEAP gene, levels of enzymatically active SEAP were measured in the CSF, blood serum, and in brain extracts. RESULTS: Intrathecal spinal canal delivery of lentivectors to newborn mice resulted in patchy, widely scattered areas of beta-gal expression mostly in the meninges. The transduction of the meningeal cells was confirmed by in situ PCR. Following the spinal infusion of a lentivector carrying the SEAP gene, sustained presence of the reporter protein was detected in the CSF, as well as in blood serum, and brain extracts. CONCLUSIONS: These findings indicate that intrathecal injections of lentivectors can provide significant levels of transgene expression in the meninges. Unlike intracerebral injections of lentivectors, intrathecal gene delivery through the spinal canal appears to produce a wider diffusion of the transgene. This approach is less invasive and may be useful to address those neurological diseases that benefit from the ectopic expression of soluble factors impermeable to the blood-brain barrier.     

Intranuclear dynamics of DNA polymerase alpha differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Intranuclear dynamics of DNA polymerase α differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase α activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase α activity, with the majority (60–75%) localized in soluble nucleoplasm and a significant amount (13–20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase α was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60–80%) of DNA polymerase α activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60–80%) of DNA polymerase β activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase α molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase α which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase β in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

A New Brain Drug Delivery Strategy: Focused Ultrasound-Enhanced Intranasal Drug Delivery

Central nervous system (CNS) diseases are difficult to treat because of the blood-brain barrier (BBB), which prevents most drugs from entering into the brain. Intranasal (IN) administration is a promising approach for drug delivery to the brain, bypassing the BBB; however, its application has been restricted to particularly potent substances and it does not offer localized delivery to specific brain sites. Focused ultrasound (FUS) in combination with microbubbles can deliver drugs to the brain at targeted locations. The present study proposed to combine these two different platform techniques (FUS+IN) for enhancing the delivery efficiency of intranasally administered drugs at a targeted location. After IN administration of 40 kDa fluorescently-labeled dextran as the model drug, FUS targeted at one region within the caudate putamen of mouse brains was applied in the presence of systemically administered microbubbles. To compare with the conventional FUS technique, in which intravenous (IV) drug injection is employed, FUS was also applied after IV injection of the same amount of dextran in another group of mice. Dextran delivery outcomes were evaluated using fluorescence imaging of brain slices. The results showed that FUS+IN enhanced drug delivery within the targeted region compared with that achieved by IN only. Despite the fact that the IN route has limited drug absorption across the nasal mucosa, the delivery efficiency of FUS+IN was not significantly different from that of FUS+IV. As a new drug delivery platform, the FUS+IN technique is potentially useful for treating CNS diseases.     

Transpapillary Drug Delivery to the Breast

The study was aimed at investigating localized topical drug delivery to the breast via mammary papilla (nipple). 5-fluorouracil (5-FU) and estradiol (EST) were used as model hydrophilic and hydrophobic compounds respectively. Porcine and human nipple were used for in-vitro penetration studies. The removal of keratin plug enhanced the drug transport through the nipple. The drug penetration was significantly higher through the nipple compared to breast skin. The drug’s lipophilicity had a significant influence on drug penetration through nipple. The ducts in the nipple served as a major transport pathway to the underlying breast tissue. Results showed that porcine nipple could be a potential model for human nipple. The topical application of 5-FU on the rat nipple resulted in high drug concentration in the breast and minimal drug levels in plasma and other organs. Overall, the findings from this study demonstrate the feasibility of localized drug delivery to the breast through nipple.     

Genetic manipulation of individual somatic mammary cells in vivo reveals a master role of STAT5a in inducing alveolar fate commitment and lactogenesis even in the absence of ovarian hormones

Assessing the molecular control of development and cell fate in individual cells in the intact mammary epithelium has not been possible to date. By exploiting an intraductal retrovirus (RCAS)-mediated gene delivery method to introduce a marker gene, we found that ductal epithelial cells are turned over with a half time of approximately 1 month in adult virgin mice. However, following RCAS-mediated introduction of a constitutively activated STAT5a (caSTAT5a), caSTAT5a-activated ductal epithelial cells expand and replace other cells in the epithelium, eventually forming a mammary gland resembling that in a late pregnant mouse, suggesting that STAT5a activation alone is sufficient to mediate pregnancy-induced mammary cell expansion, alveolar cell fate commitment, and lactogenesis. Furthermore, such caSTAT5a-induced alveolar differentiation does not require ovarian functions, although caSTAT5a-induced cell proliferation is partly reduced in ovariectomized mice. In conclusion, in this first report of studying the developmental role of a gene in a few cells in a normally developed virgin mammary ductal tree, STAT5a activation causes alveolar fate commitment and lactogenesis, and with the help of ovarian hormones, drives alveolar expansion.     

Gene transfer by jet injection into differentiated tissues of living animals and in organ culture

Jet injection can be used to introduce genes into the cells of differentiated tissues of living animals and organ cultures. When a solution of plasmid DNA is jet injected into a selected tissue or organ, cells lying in or near the path of the jet injection are transfected with the DNA and the introduced gene(s) are expressed. Since there is minimal morbidity from each jet injection, multiple injections can be performed at the same or nearby sites. Both mRNA and protein expression from transfected genes can be quantitated using standard methods. In addition, the technique is an efficient means of DNA immunization. Methodology for using jet injection to transfer plasmid DNA into the cells of skin, fat, mammary gland, and muscle are described.     

Enhanced Expression of Adenovirus Encoding rhEPO Assisted by BAPTA

Infection efficiency is the key issue for gene delivery using adenovirus vector and usually unsatisfactory. In this study, recombinant adenoviruses encoding recombinant human EPO were prepared using the Adeasy system, and injected into the mammary gland of goats via the teat canal. BAPTA was used to treat the mammary gland to facilitate adenoviruses infection compared with EGTA. Milk serum was collected from the infected mammary gland and characterized by ELISAs and Western blotting. Expression level of rhEPO from the group treated by BAPTA was higher than that treated by EGTA.     

Use of fluorescent microspheres to localize in vivo gene transfer injection sites

The potential for using gene therapy to treat a variety of disease states is growing rapidly. Many vector types and delivery systems have been developed that allow the optimization of protein production levels and kinetics for a given therapeutic gene product. In cases in which a transient, localized delivery of gene product is desired, any determination of the locale of transfected tissue by non-marker genes is problematic. We describe a technique by which the use of fluorescent microspheres can help in identifying potentially transfected tissue. Adenovirus containing the gene for beta-galactosidase (beta-gal) was mixed with fluorescent microspheres and injected into rat skeletal muscle and porcine myocardium. The injection sites could be visualized under ultraviolet light and correlated with beta-gal enzyme expression. This method is simple, inexpensive and generally useful for in vivo gene transfer experiments.     

Antibody-Guided In Vivo Imaging for Early Detection of Mammary Gland Tumors

BACKGROUND: Earlier detection of transformed cells using target-specific imaging techniques holds great promise. We have developed TAB 004, a monoclonal antibody highly specific to a protein sequence accessible in the tumor form of MUC1 (tMUC1). We present data assessing both the specificity and sensitivity of TAB 004 in vitro and in genetically engineered mice in vivo. METHODS: Polyoma Middle T Antigen mice were crossed to the human MUC1.Tg mice to generate MMT mice. In MMT mice, mammary gland hyperplasia is observed between 6 and 10 weeks of age that progresses to ductal carcinoma in situ by 12 to 14 weeks and adenocarcinoma by 18 to 24 weeks. Approximately 40% of these mice develop metastasis to the lung and other organs with a tumor evolution that closely mimics human breast cancer progression. Tumor progression was monitored in MMT mice (from ages 8 to 22 weeks) by in vivo imaging following retro-orbital injections of the TAB 004 conjugated to indocyanine green (TAB-ICG). At euthanasia, mammary gland tumors and normal epithelial tissues were collected for further analyses. RESULTS: In vivo imaging following TAB-ICG injection permitted significantly earlier detection of tumors compared with physical examination. Furthermore, TAB-ICG administration in MMT mice enabled the detection of lung metastases while sparing recognition of normal epithelia. CONCLUSIONS: The data highlight the specificity and the sensitivity of the TAB 004 antibody in differentiating normal versus tumor form of MUC1 and its utility as a targeted imaging agent for early detection, tumor monitoring response, as well as potential clinical use for targeted drug delivery.