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A transgenic mouse model for SCLC: Expression of Hel-N1 in mouse lung

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A transgenic mouse model for SCLC: Expression of Hel-N1 in mouse lung

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Content A TRANSGENIC MOUSE MODEL FOR SCLC: EXPRESSION OF HEL-N1 IN MOUSE LUNG by Taian Chen A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SPUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree Master of Science (BIOCHEMISTRY) May 2001 Copyright 2001 Taian Chen Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY O F S O U T H E R N CALIFORNIA THE GRADUATE S C H O O L UNIVERSITY PARK LOS ANGELES. CALIFORNIA S 0 0 0 7 This thesis, written by .JSi /H im ________ under the direction of h..s.St.~Thesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of The Graduate School, in partial fulfillment of the requirements for the degree of & V I f .ir^ ....— —— — Dess D ate....m ..lkuJQ Q l.____ THESIS CQMMI1 It, Ckmrmas Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents Introduction 1 Material and Method 9 Result 20 Discussion 26 Figures 30 Reference 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures Figure la Not I Site Knockout of pKS-CCIO Plasmid Figure lb G954 Double Digests to Show Not I site Removal Figure lc pKS-mCCIO Plasmid Map Figure 2a Oligo-linker Insertion Orientation Figure 2b H I7 Plasmid Map Figure 3a Hel-Nl Gene Insertion Figure 3b H33 Digests of Enzymes used for Transgene Isolation Figure 3c H33 Plasmid Map Figure 4 PCR analysis of founder candidates (IA 13 - 19) Figure 5 Southern blot analysis of founder Figure 6 Southern Blot of Trangenic Progeny Figure 7 IA 17 Transgenic Family Tree Figure 8 Western blot analysis of mice lung and brain Figure 9 RT-PCR of Transgenic Mice Lung RNA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction Lung cancer has been the leading cause of cancer death in men since early 1950s, and the rate has been dramatically increasing every decade until 1990, when it peaked at 75 deaths per 100,000 males. (1) However, in female, it wasn’t until the mid 80s when lung cancer finally surpassed breast cancer as the leading cancer causing death, and is not showing any signs of reversing the trend. 5-year survival is less than 15% for all kinds of lung cancer. In the U.S. alone, over 158,000 people die each year just from lung cancer, a statistic comparable to having one year worth of traffic accident deaths in the state of California (according to 1998 statistic) happening in one week, and continue for a whole year. Almost 90% of lung cancers are caused by cigarette smoke, therefore it is imperative that programs for smoking prevention and cession be implemented. The only true way to prevent lung cancer is to just stop smoking. However, it takes about 20 years for lung cancer to develop, so even in the event that every smoker in world permanently stops smoking today, lung cancer would still occur for a long time to come. If a smoker stops smoking, the risk of lung cancer decreases very slowly over time, and remains high for many years. The combination of the long development time and slow decrease of risk means that lung cancer is still likely to be the leading cancer causing death for many years to come. Due to the multiple cell types involved, lung cancer is considered a heterogeneous disease. There are four major kinds of lung cancer: squamous cell 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. carcinoma (-30%), adenocarcinoma (-30%), large cell carcinoma (-20%), and small cell carcinoma (-15 - 20%). The most aggressive and metastatic of all lung cancers is small cell lung carcinoma (SCLC) which has neuroendocrine characteristics. SCLC is second most metastatic of all cancer, following only pancreatic cancer. The 5-year survival of SCLC patient is only 5% for all stages, and a meager 1.2 % for distally metastasized tumor. SCLC derives its name from the tumor cells that are characterized by the small size. The cells have a round to fusiform shape, scant cytoplasm, finely granular nuclear chromatin, and absent or inconspicuous nucleoli. One reason that accounts for the low 5-year survival rate of SCLC is the inability to diagnose this cancer early. Most patients are beyond the initial stages at time of diagnosis, thus making treatment less effective. Moreover, local treatments such as radiotherapy or surgery are not effective in prolonging survival beyond a few weeks. (13) The cause of death in most instances is systemic metastases. Due to that factor, the current most effective therapy is chemotherapy. Even so, most chemotherapy treated SCLC patients eventually develop recurrent disease, and maintenance chemotherapy has no effect on survival rate. The survival of SCLC patients has barely increased over the last 20 years (24), and this is due to a lack of new approaches for the treatment of SCLC and poor understanding of the molecular basis of the disease. An added problem making it hard to develop new treatment approaches or to discern the molecular basis of SCLC is the absence of an animal model for SCLC. The lack of an animal model isn’t due to a lack of interest or effort. The initial 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. approach to making an animal model was to expose animals to toxins and other factors that are known to cause lung cancer in human, such as cigarette smoke and increased CO2. That method did yield animal model of dog, rabbit, and hamster for lung adenocarcinoma, but not SCLC. (19) Such exposure protocols contain a lot of unknown and uncontrollable factors within the system, and therefore are not ideal systems to study the molecular basis for the disease. An alternative method for getting an animal model would be through genetic manipulation to induce the expression of cancer-causing gene in lung, i.e. transgenic mice. With transgenic models, one could target specific cell types to overexpress or knockout specific genes and see the effect of such changes. With a promoter specific for the right cell type in lung may be the key to SCLC animal model. So what is the right cell type in lung for SCLC? In order to develop cancer, the cell must have the ability to grow, such as epithelial cell. There are four different types of epithelial cells in adult lung tissue: non-ciliated Clara cells, ciliated cells, type I, and type II cells. Pulmonary endocrine cells (PNE), a group of cells distributed within the mucosa, were believed to have a role in lung development, because they are present at the highest level just before birth and drop off postnatally (23). Since SCLC have neuroendocine characteristics, PNE cells were first thought to be the possible precursor cells of SCLC. However, studies (16) showed that induction of PNE cell hyperplasia by treating hamsters with the carcinogen, revealed that the epithelial cells immediately adjacent to the PNE cells were affected, and not the actual PNE cells themselves. This finding concluded that PNE cells actually 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. arise from differentiation of the adjoining airway epithelium cells, Clara cells (or their lineage). From the studies, it seemed like Clara cells are the very likely the precursor cells for lung cancer. Clara cells are the non-ciliated secretory cells of the pulmonary epithelium characterized by a large apical dome shape and abundant endoplasmic reticulum. The cells secrete a few proteins including the SP-A, SP-B, SP-D, leukocyte protease inhibitor, a trypsin-like protease, and a 10 kD protein known as the CC10. The CC10 protein has functions involved in lung repair and inflammatory response. By using Clara cell specific promoter, one may be able to get lung specific expression of gene, and possibly SCLC development. Other laboratories have used transgenic expression of oncogenes targeted to Clara cells as well as other cell types but none of these approaches (summarized in the next §) led to the development of SCLC. One population of cells that has been targeted in the past is the PNE cells. To get PNE cell specific expression, the calcitonin gene-related peptide (CGRP) was used. (3) The promoter was attached with either full-length oncogenes c-myc or Simian Virus 40 Large T Antigen (SV40 TAg). c-myc overexpression in transgenic mice has been found to cause tumor formation. SV40 TAg binds the tumor suppressor protein pRb or p53, and results in unregulated cellular growth. The CGRP/c-myc mice never developed any tumors despite high level of c-myc expression. The CGRP/SV40 TAg mice only developed thyroid cancer and died quickly before analysis could be performed. Thus CGRP promoter was not a good choice for our experiment. 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Another population of cells that have been targeted is the Type II epithelial cells lining the alveoli. A transgenic model was generated using surfactant protein C (SP- C) promoter with SV40 TAg. (27) The transgenic SP-C/ SV40 TAg mice developed pulmonary tumors that were morphologically identical to adenocarcinomas. In this study, CC10 mRNA was detected in the majority of pulmonary tumors. SP-C mRNAwas also detected, but at much lower frequency. The presence of these mRNA showed that both type II and Clara cells are involved in the tumorigenesis observed in the mice. Transgenic models using Clara cells as the target have also been made. Either the CC10 promoter (18) or the rabbit uteroglobin promoter (22) was linked to SV40. Uteroglobin is also a marker protein for Clara cells, just like CC10. In both models, the mice developed multifocal adenocarcinoma of the lung, but neither developed SCLC. Thus, so far, attempts at overexpressing oncogenes have generated lung cancer, but not SCLC. Of the previous transgenic models, the CGRP model died from thyroid cancer before lung cancer could develop. Use of the SP-C promoter gave lung cancer, but expression was targeted to type II cells which are located at the periphery of lung. SCLC usually occurs more proximally, in the larger air pathways. CC10 promoter seemed to be the best suited for our purpose. However, that model only developed adenocarcinoma and not SCLC. In order to get an animal model of SCLC, another gene would be chosen for overexpression which has the ability to 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. push Clara Cells toward neuronal differentiation. A minor percentages of SCLC patients were found to have paraneoplastic encephalomyelitis sensory neuronopathy (PEM/PSN), a neurologicaldisorder. (26) Patients show signs of dementia, cerebellar degeneration, brain stem encephalopathy, myelopathy, and sensory neuropathy. The disease is caused by an autoimmune response that is primarily directed against the SCLC and the Hu protein it produces, which is normally a neuronal protein. Hu proteins are RNA binding proteins that show homology to the essential Drosophila protein ELAV (embryonic lethal and altered visual), around 40% at nucleotide level. The proteins within the family are 35 - 40 kD and contain three highly conserved RNA recognition motifs that are known as RRM1, RRM2, and RRM3. For Drosophila, ELAV is required for normal development and maintenance of the fly nervous system. Those that lack ELAV do not survive embryonic development while those that carry mutated ELAV exhibit a disorganized nervous system. Thus far, four different Hu proteins have been identified in humans: HuD, Hel-Nl (HuB), HuC, and HuR. HuR, the least conserved among the four, is ubiquitously expressed in all cell types while the other three proteins are primarily expressed in neurons. Hel-Nl is also weakly expressed in the testes and ovaries. Hu proteins were discovered originally because they were the targets of antibodies found in patients with PEM/PSN associated with SCLC. Anti-Hu antibodies from patients have been shown to recognize with HuD, HuC, and Hel-N 1 expressed by neurons and SCLC. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Specific Hu genes are expressed in a hierarchy during neurogenesis. Hel-Nl is induced first in very early postmitotic neurons, followed by HuD and HuC. In adults, the proteins are expressed in various regions of the nervous system. Hel-N 1 detected in SCLC is not endogenous. It has been shown that Hel-Nl is expressed in SCLC using RT-PCR. The promoter elements of Hel-Nl suggested that they were likely to contribute to the neuronal specificity of Hel-Nl mRNA expression. Previous work done in our lab also showed that Hel-N 1 was expressed in all 10 SCLC lines tested. (4) All Hu family proteins, including Hel-Nl, bind tightly to AU- rich sequence. This has been shown to cause mRNA stabilization in certain cases. A study showed that Hel-Nl could bind to transcript containing the 3’ UTR of c-myc and c-fos mRNA. (15) The ability of Hel-Nl able to bind to c-myc mRNA is potentially interesting because c-myc levels was known to be elevated in SCLC. In experiment done previously in our lab, it’s been shown that myc and fos has been stabilized in many SCLC cell lines. Thus, Hel-Nl may serve both as a stabilizer of oncogene mRNA as well as making Clara Cell differentiate toward neuronal pathway. Moreover, CClO/Hel-Nl mouse and CC10/SV40 TAg, which was already made by Dr. DeMayo, cross would represent three most common phenomenon observed in SCLC: myc amplification (40%), p53 deletion (80%), and pRB (90%) deletion. By generating a transgenic animal model of SCLC, some of the molecular changes of the disease may be easier to discuss. In most human SCLCs, deletion of chromosome 3p is almost always observed. It is still unknown what caused this 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phenomenon, and the biochemical and molecular effects of this deletion which leads to the onset of SCLC. The identity of the deleted gene(s) is still a mystery. Many other molecular questions concerning SCLC could be answered much easier if an animal model was available. By having an animal that is prone to develop SCLC, researchers could see the step by step changes of the lung morphology until the onset of cancer. Other cell markers which may have elevated levels right before cancer and declined afterward could be identified. This can lead to earlier detection methods. Moreover, better treatment approach could be developed if an animal model of the disease was available for study by testing different treatments on SCLC animals for the most effective one. 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Material and Method Transgene Plasmid Construct pKS CC10 plasmid construct obtained from Dr. Franco DeMayo contained the 0.8 kb Clara Cell 10 lung specific promoter, rabbit beta globin intron, and the human growth hormone polyadenylation signal. The plasmid arrived in forms of filter paper and was soaked in 100 |ll of TE. 4 |il of the TE containing plasmid were transformed into 20 p .1 of DH12S competent cells, heat shocked for 37 °C for 1 minute and placed on ice. 200 pi of LB was added to the cells and incubated at 37 Q C for 1 hour then plated on ampicillin LB agar plates. The plate was incubated overnight at 37 °C and one colony was selected for growth in 200 ml of LB. The plasmid was finally obtained by Qiagen Maxi Prep Kit. Because we wished to use a Not I site for later cloning steps, the Not I site upstream of promoter had to be removed. pKS CC10 was linearized with Not I overnight at 37 °C then extracted and precipitated by phenol/chloroform method. Blunting of Not I site was done by adding dNTP mixture (10 mmol/p.1) and T4 DNA polymerase to the linerized plasmid. The blunting mixture was incubated at 12 °C for 30 minutes then the concentration was estimated by running on an agarose gel. 50 ng of the blunted DNA was self ligated with T4 DNALigase overnight at 16 °C, transformed, and streaked onto an Amp plate. Colonies that grew from the overnight plateing were used for mini prep. An autoclaved toothpick was used to pick each individual colonies and then dropped into a test tube with 2 ml of LB containing 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ampicillin. The plasmid was allowed to grow in LB by placing the test tubes on a spinning rack at 37 °C overnight. The clones were mini prepped. (20) 5 pi of mini prep DNA was digested for 2 hours with Not I enzyme to see if the Not I site had been removed. Although the EcoR I site downstream of rabbit beta globin is ideal for cloning in the gene of interest, a Not I site downstream of EcoR I site is required because Hel-N 1 coding sequence was to be isolated by EcoR I and Not I digest. Thus before Hel-Nl, the gene of our interest can be cloned in, an oligonucleotide linker need to be inserted for the purpose of generating a Not I site 3 ’ of EcoR I site. Oligonucleotides were ordered from Fisher Scientific Genosys. The oligo EcoNot Top (AAT TCT CTT TCC TTG CGG CCG C) and EcoNot Bottom (AAT TGC GGC CGC AAG GAAAGA G) were reconstituted into concentration of 100 pmol / pi with TE. In order to generate the oligonucleotide linker, the two oligonucleotides had to be annealed together. 10 pi of each diluted oligonucleotide was mixed, placed in a 80 °C heat block for 1 minute, and was then allowed to cool to room temperature before usage. 5 pg of pKS CC10 plasmid clone (with Not I site removed) was digested with EcoR I overnight at 37 °C heat block. The linearized plasmid was run on preparatory agarose gel, and then extracted with Qiagen Quick Spin Gel Purification Kit. The purified DNA was ligated with the annealed oligonucleotide linker with a molar ratio of 1:10. The ligation was incubated in 16 °C heat block overnight. The control did not have linker added to the reaction mixture. Following incubation, 4 pi of ligation 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mix was transformed into DH12S competent cells (as described above) and then streaked onto ampicillin LB agar plate and incubated at 37 °C overnight. Colonies were innoculated for mini preps. DNA obtained from the mini preps was digested overnight in 2 separate reaction tubes with one tube containing Sph I / Not I enzymes and the second tube Sph 11 EcoR I enzymes. Restriction digests were run on 1% Metaphore gel (BMA) to determine whether the oligonucleotide was inserted, and if so, in which orientation. To further ensure that the right clone has been obtained, the potential clone was sequenced by Norris Cancer Center Core Sequencing Facility. The oligonucleotide used for sequencing was ordered from Fisher Scientific Genosys. The primer (ATG GGTTCT TCT TTTTCC TAC AG), CC10 Sequencer, is located 5’ of EcoR I site. The annealing temperature of the oligonucleotide was 59 °C. The gene of interest, Hel-Nl, needed to be isolated from pET plasmid clone, F622 (Ite Laird-Offringa Lab). F622 was digested with both EcoR I and Not I overnight with excess enzyme. The digest was run on a 1% agarose gel for 1 hour at 120 volts and the 1.2 kb EcoR I /Not I fragment was purified by Qiagen Quick Spin Gel Purification Kit. Because both Hel-Nl and anchor fragments were the same length, both were isolated from this digest. To prevent excess loss of DNA from further purification process, all the 1.2 kb fragments isolated from the purification were used in ligation. The oligonucleotide linker inserted pKS CC10 plasmid was digested overnight with EcoR I and Not I at 37 °C heat block. The linearized plasmid was run on 1% agarose gel for 1 hour at 120 volts and then extracted by 1 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Qiagen Quick Spin Gel Purification Kit. The purified DNA was ligated with the 1.2 kb fragment isolated previously from F622 with a molar ratio of 1:5. The ligation was incubated in a 16 °C heat block overnight. The control did not have 1.2 kb fragment (Hel-Nl/Gene III) added to the reaction mixture. The ligation mixture was same as above and colonies were pick for mini prep. DNA obtained from the mini preps was digested overnight with Bam HI (an unique site on both the pKS CC10 vector and the anchor fragment) for the purpose of differentiating whether the insertion was Hel-Nl or Gene III. To ensure the right clone was obtained, the clone was sequenced by the Norris Cancer Center Core Sequencing Facility using the CC10 Sequencer primer. Transaene Isolation and Purification For the purpose making transgenic mice, a fragment containing the CC10 promoter, rabbit beta globin intron, and Hel-Nl had to be isolated from the plasmid. 10 |ag of pKS-CCIO with Hel-Nl insertion (H33) was digested with ApaL I overnight at 37 °C. The digest was run on 1% agarose gel at 120 volts for 1 hour, and the 4.6 kb fragment was purified by Qiagen Quick Spin Gel Extraction Kit. The isolated 4.6 kb fragment was digested overnight with both Fse I and Cla I. A 2.9 kb fragment was purified by gel purification. The multi-step isolation process was utilized to ensure that incomplete digests or other contaiminations would not affect the purity of the transgene. The 2 jjlI of purified fragment was run on a 1% agarose 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. gel for 1 hour at 120 volts to estimate the concentration. 2 fig of the gel purified fragment was further purified by Elu-tip (Schleicher & Schuell). The 2.9 kb fragment contained the CC10 promoter, intron, Hel-Nl coding sequence, and human growth hormone stop codon, and was microinjected into fertilized egg during micro injection process. (Norris Transgenic Core; see below) Starting and Maintenance of Transgenic Mice Colony (Trot# 9723) For the purpose of microinjection of the trangene, 24 FvB/N (Friendly Virus B/Albino) wildtype mice were ordered from Jackson Lab. The males were 7 weeks old, and the females were 4 weeks old. The optimal age for superovulation in female mice is around the age of 3 - 4 weeks. Animals were housed in the Vivarium in Norris Cancer Center from the time of arrival. All animals and the progenies were housed in the Norris Cancer Vivarium and strictly adhere to the rules within. The temperature was kept constant at between 69 °C and 72 °C, as was humidity. The light within the vivarium dim at 5:30 pm and was complete dark at 6:00 pm. Cage changing was done twice in a week. Once removed from the facility, an animal can no longer be put back in. All progenies were weaned at approximately 3 weeks of age, unless the animal was too small in 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. physique. If an animal was to be dissected for analysis, it was euthenized by CO2 beforehand. For RNA and protein expression experiments, different tissue samples were obtained from both wildtype and transgenic animals, including: lung, heart, brain, liver, uterus, kidney, ovaries/testis, and spleen. All surgical equipment were cleaned by ethanol and then flaming over fire after the alcohol has evaporated. The postmortem animal was disposed of by the vivarium. Genotvping by PCR For the purpose of identifying transgenic mice, genomic DNA from mice was necessary for genotyping by either polymerase chain reaction (PCR) or Southern Blot. Genomic DNA from animal was obtained by tail biopsy. (14) When the animal had reached the age of three weeks, the tip of tail (around 1 cm or less) was cut. The tail section was directly transferred to a tube containing 500 pi of lysis buffer (100 mM Tris HC1, 5 mM EDTA, 0.2 % SDS, 200 mM NaCl, 100 jig Proteinase K/ml). The tube was incubated at 50 °C on rocker overnight or until the tissue has completely dissolved. Spinning for 10 minute at 13,000 rpm anchored the remaining hairs to the bottom of the tube, and the aqueous layer was transferred to another tube containing 500 ml of isopropanol. By vigorous shaking of tube, a white DNA pellet was precipitated. A toothpick or pippette tip was used to fish out the pellet and transfer it to another tube with 150 pi of TE for reconstituting. Place Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the tube in the 50 °C rocker to aid in dissolving the DNA. For PCR analysis, an aliquot of previously obtained genomic DNA was diluted 50 times in TE, and 5 jjl was used. The 5’ primer used for PCR analysis was CC10 Sequencer and the 3’ primer used was Hel-Nl SEQ3 (CGA TGC AAT TTC CTC ATT TTA TTA G) ordered from Fisher Scientific Genosys. The 5’ primer was designed to sit in the intron upstream of the Hel-Nl cDNA and the 3’ primer hybridized to the human growth hormone sequence containing the poly (A) signal following Hel-Nl. Only a transgenic animal containing the injected transgene would give a product of 1.3 kb. PCR screening was done with 10X buffer (Promega), MgCE, 25 mM of each nucleotide (dATP, dCTP, dTTP, dGTP), and 100 pmol of each primer. The PCR was performed using a DNA Engine (MJ Research) with the following conditions: 1. 95 °C for 3 minutes; 2. 92 °C for 1 minutes; 3. 55 °C 1 minute; 4. 72 °C for 2 minutes. Step 2 to 4 was repeated for 30 cycles, then the samples were kept at 4 °C for storage. 25pl of DNA product was run on a 1% agarose gel for 45 minutes at 120 volts. PCR screening of CC10 promoter was done with primers H33CC105’ (CCT GGG AAT GGC TAA CTA CTT G) and H33CC103’ (TAT TGC AAG TTG GCC GAT TAG AC). The 5’ primer hybridized to the sequence that is just downstream of the Fse I restriction site that was used to isolate transgene and the 3’ primer anneald to a specific sequence in rabbit beta globin. 25jil of DNA product was run on a 1% agarose gel for 45 minutes at 120 volts. 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Genotvping bv Southern Blot For Southern Blot analysis, 5 -1 0 pg of genomic DNA was digested overnight with either Bam HI or EcoR I in 50 pi volume. The digest was run on a 0.8% agarose gel at 40 volts until the loading dye had reached the bottom of the gel. The gel was treated with 0.2 M HC1 with gentle shaking twice for 15 minutes and then rinsed thoroughly with water at least four times before denaturation. The gel was soaked in denaturation solution (1.5 M NaCl, 0.5 M NaOH) for at least 45 minutes, and then rinsed with double distilled water twice to remove the NaCl. Lastly, neutralization solution (1.5 M NaCl, 0.5 M Tris Cl, pH 7.0) was added and the gel remained on the shaker for another 45 minutes. The gel was blotted overnight onto Zetabind membrane (Biorad, Hercules CA) until the transfer was deemed complete by checking for remaining DNA on the agarose gel. The DNA on the membrane is crosslinked at 1200 joules with Ultraviolet Crosslinker. (Ultra LUM) The membrane is prehybridized in a hybridization cylinder at 65 °C for 15 minutes with 10 ml of CHURCH buffer (500 mM NaPi (1 M Na2HPC>4 + 1 M NaHaPCL) pH6.8, 7% SDS, ImM EDTA). The Hel-Nl specific probe used in the experiment was the 384 nucleotide sequence of the Hel-Nl hinge region isolated by PCR with primers HuR/Hel5 ’ (GTT TAT TCG ATT TGA CA) and HuR/Hel3 ’ (CCAAAC ATC TGC CAC AGG AT) with a Hel-Nl template plasmid and then purified by gel extraction with Qiagen Quick Spin Gel Extraction Kit. 10 - 20 ng of DNA was reconstituted with water to a total volume of 37 pi. 5 pi of OLB was 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. added to the tube and then incubated in 100 °C heat block for 5 minutes. Once the tube has cooled to below 60 °C, 1 pi of BSA (10 mg/ml), 5 pi of oc-3 2 P - dCTP (3000 Ci/mmol; 10 pCi/ml), 2 pi of Klenow (New England Biolab), were added in the order listed. The mixture was incubated in a 37 °C heat block for 1 hour, then 50 pi ofTioEioSo.i (TrisiomM EDTAiomM SDSo.i%) was added to stop the reaction. The labeling solution was spun over the Sephadex G-50 column for 1.5 minute at 1500 rpm. The probe was heated for 5 minutes at 100 °C before being added to the prehybridizing column to be incubated at 65 °C overnight. The membrane was washed with 1% SDS and 2% SSC (gradually lowing the concentration of both until 0.5 % SDS and 1% SSC) at 65 °C for 15 minutes, exposed overnight in a phosphor cassette, and analyzed with Phosphorimager (Molecular Dynamics). Expression Analysis with Western Blot Western blot transfer was done to determine the expression of Hel-Nl protein in various tissue samples from both wildtype and transgenic animals. The protein was isolated by homogenizing the tissue sample with lysis buffer (1% Triton X, 300 mM NaCl, 50 mM Tris, pH 7.4) in a muscle tissue homogenizer. After homogenizing, the protease cocktail consisted of 100 mM PMSF (5 pl/ml), 1 p,g/ml Pepstatin (0.7 p.l/ml), and 1 pg/ml Leupeptin (0.5 pl/ml) was added. The homogenized content was spun at 3000 rpm for 3 minute and the supernatant containing protein was transferred to 1.5 ml tubes. The tubes were kept at -80 °C freezer until ready for analysis. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 pi fractions of the various protein elutions were run on a polyacrylamide SDS denaturing gel. The concentrations of the proteins were estimated by Coomasie blue stained gel. An estimated 10 jig of protein sample was added to 10 pi of 2x lysis mix (1M Tris pH 6.8,20% SDS, 0.5 pi of glycerol, 0.5 pi of |3- mercaptoethanol, Bromophenol blue saturated, water) before running on polyacrylamide SDS denaturing gel. The gel was transferred to nitrocellulose membrane (MSI). The primary antibody used was human anti-Hu serum from a SCLC patient and the second antibody used was goat anti human antibody conjugated with biotin. The membrane was blocked with TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.05 % Tween 20) with 3% BSA for 2 - 3 hours. The membrane was probed with 5 pi extravidin horseradish peroxidase from Sigma (HRPO) diluted in 10 ml of TBS, and incubated for 30 minutes. After incubation, the membrane was washed 6 times for 5 minutes each in TBS (10 mM Tris pH 8.0,150 mM NaCl). The membrane was then developed with Super Signal (Pierce) for 5 minutes following the manufacturer’s recommendations and exposed to X-ray film for 5 and 10 seconds. Exposure longer than 10 second results in overbumed films. Expression Analysis by RT-PCR RNA from tissue samples was extract by Trizol method (Life Tech). For reverse transcript polymerase chain reaction, 2,5 pg of RNA was used from each sample. 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The total volume of the RNA sample was brought up to 25 pi with DEPC-H2O. The reverse transcription was done with 2.5 pi MMLV-RT (Gibco), 0.2 pi BSA, 5 pi dNTP mixture (25 mM each dNTP), 3 pi of 3’ primer, 1.25 pi RNAsin, and 5 pi of DTT. A negative control was also prepared using all the same reagents but missing MMLV-RT. The RT reaction PCR program was 10 minutes at 22 °C, 45 minutes at 42 °C, 3 minutes at 90 °C, cool to 0 °C and add 1.25 pi of fresh RT to each tube, 45 minutes at 42 °C, 10 minutes at 75 °C. The cDNA samples were stored at -20 °C until usage. 2.5 pi of each cDNA sample was used in subsequent PCR with Helhinger5’ (AAA GGC CTAAAT GGC CAG AAA) and Helhinge3’ (CAG GAG CCA GGT TGT ACA C) from MGW Scientific. The primers hybridized to the hinge region of Hel-Nl and would give Hel-Nl specific PCR products. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results Construction and Analysis of pKS CC10 containing human Hel-Nl Coding Sequence Because the Not I site upstream of CC10 promoter sequence would interfere with Hel-Nl insertion afterward due to a Not I site on the Hel-Nl cDNA, it was eliminated by blunting and then religation. Fig. la (G954 x Not I) shows that G954 Was running similar plasmid, that the unique Not I site had been removed. Subsequent double digests of G954 with Not I and one other unique enzyme (Fig. lb) showed a linearized plasmid, demonstrated that Not I site not longer existed. In order to clone in the human Hel-N 1 sequence, a Not I site need to be generated downstream of EcoR I, therefore an oligonucleotide was inserted for that purpose. If the linker had been inserted in the proper orientation, Not I/Sph I double digest would yield a 236 nucleotide fragment, and EcoR l/Sph I double digest would yield a 252 nucleotide fragment. By running the digests on a Metaphore gel, the orientation of the clones can be differentiated. Fig. 2a (H17 x Not l/Sph I and EcoR l/Sph I) shows that HI7 was the clone with the proper orientation linker insertion. HI 7 was also sequenced to confirm what was seen on gel. The human Hel-Nl coding sequence was isolated from F622 by EcoR I and Not I double digest. The 1.3 kb fragment from the digest was purified and ligated with vector HI7, which had also been digested with EcoR I and Not I. Bam HI was a 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. unique restriction enzyme site on HI7, and also found on Gene III sequence, but not on Hel-N 1. Clones were digested with Bam HI to check whether Hel-N 1 or Gene III had been inserted. Fig 3a (H23-34) shows that H33 was linearized and was running at a higher position than the control (HI 7 x Bam HI), suggesting that there was Hel- Nl insertion. H33 was also digested with Sph I, Cla I, and Fse I to show that it was the predicted size of 5.9 kb length, as well as a EcoR I /Not I digest to show the Hel- Nl cloned in was the same length as the fragment previously isolated from F622. H33 was also sequenced to confirm that existence of Hel-Nl within the plasmid with primer CC10 sequencer. Transgenic Founders From the injection of transgene into approximately 70 fertilized eggs of FvB/N mice, 6 mouse pups (IA 13 - 18) were bom, 3 males and 3 females. Tails of the pups were cut at 3 weeks of age, at the same time they were weaned. DNA was extracted and genotyping was done by both PCR and Southern Blot analysis. The 5’ primer (CClOSequencer) is complimentary to sequence in beta globin intron, hybridizing a few nucleotides upstream of the Hel-Nl coding region. The 3’ primer anneals to the polyadenylation signal downstream of the Hel-N 1. If a 1.3 kb band is observed as a PCR product, that sample must contain the transgene. Analysis of the 6 pups’ DNA samples showed that two of the female mice, IA 17 and IA 18, contained the transgene (Fig. 4 lane 8 and 9 white arrow). The experiment was repeated with only 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IA 17 and IA 18 samples to ensure there wasn’t any cross contamination when the samples were loaded in adjacent lanes. Although IA 18 seemed to give a stronger signal than IA 17, DNA samples were not quantitated before PCR, therefore the result did not indicate the relative number of transgenes inserted. The PCR indicated that transgene had been inserted, but didn’t provide the information about the integrity of the promoter. To obtain this information, a PCR was performed with primers H33CC105’ and H33CC103’. The 5’ primer annealed to the Fse I site upstream of the promoter sequence and the 3’ primer bound to a sequence near the end of intron. The PCR product would span across the promoter and intron region of the transgene. PCR screening indicated that the promoter was intact in at least some copies of the insert. However, since the 2 PCR tests did not bridge the whole transgene fragment, it was not possible to know whether an intact copy of the complete transgene was inserted. To confirm the PCR results, 10 jig of DNA from all the pups were digested individually with EcoR I and Bam HI, both being unique in the trangene. When probed with a 384 nucleotide fragment of Hel-N 1 coding region, IA 17 and 18 were again shown to be positive (Fig 5). In comparing the intensity of the signals in lane 11 and 12, there seemed to be a 6 fold difference between the two samples. Based on the intensity of the signal at ~ 3 kb band (Phosphorimager analysis), IA 17 was estimated to contain 2 copies of the transgene while IA 18 was thought to carry 10 or more copies (Fig 5 lane 11 and 12). When multiple copies of a transgene have been inserted into genomic DNA, the fragments may be linked in head - t o - head, head - 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to - tail, tail - t o - tail orientations, or random combinations. Since the EcoR I site was right in the middle of transgene, all linkage formations would lead to ~ 3 kb band signal. The two higher bands were presumed to be the flanking copies of the linked transgenes. The multiple band signals seen in lane 6 were most likely due to incomplete digest of the DNA since same pattern was not observed in lane 12. Transgenic Germline Establishment Both transgenic animals appeared to be healthy and were mated to wildtype FvB/N males at week 8 obtain transgenic progenies. Both female mice (IA17 and IA 18) were able to produce healthy litters. In the second litter that IA 17 produced, 1 female pup (IA29) contained the transgene (Fig 6). Since then, all the Fi generation mice (IA 17 and 18 being Fo) transmitted the transgene at -50%, indicating that the IA17 line has established germline transmission of the transgene. That particular transgenic line has produced both male and female transgenic animals and seemed to be in full health to date (oldest Fi being 7 months old). IA 18 did not produce any pup that indicated the presence of transgene within the DNA by either PCR or Southern Blot analysis. Protein and mRNA Expression Although we knew by PCR and Southern Blot that the transgene was present in 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the DNA of the transgenic animals, those experiments did not tell us whether the gene was expressed and whether its expression was specific to the lung. Inducing Hel-Nl expression in the lung was the ultimate goal for making these transgenic lines, therefore a Western Blot analysis was performed. A female transgenic progeny of IA 29 was sacrificed and the lung and brain protein extracted. The same experiment was also performed on a wildtype female sibling of the sacrificed mouse. Fig 8 shows that when probed with human anti Hu serum, both the wildtype and transgenic neuronal protein samples contained Hu proteins. The human anti Hu serum was obtained from a SCLC patient and it reacts with all four Hu proteins, but reaction with HuR was much weaker. That result from Fig 8 was expected because Hu proteins are neuronally expressed, except for HuR which was ubiquitously expressed. On the film, the wildtype lung protein sample did not have a band seemed to indicate that the serum did not react well with HuR, as confirmed by experiment done previously in our lab. There was a band in the transgenic lung sample (Fig 8, arrow), indicating that presence of Hu protein in transgenic lung was detected. Although the transgenic lung and neuronal proteins were in adjacent lanes, there was very little chance, if none, of contamination due to the deep wells (half inch) of the gel. Recombinant Hel-N 1 protein was loaded in the middle of gel as a positive control. The control ran at a high position in the gel presumably due to the presence of the myc and his tags that are missing in native proteins. This experiment, strongly suggested that we had been able to generate a transgenic animal that expressed of the neuronal protein, Hel-Nl, in lung, driven by the a lung specific 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CC10 promoter. Although we knew the protein was being expressed from the Western Blot, RT- PCR was performed to ensure the presence of Hel-Nl mRNA. The PCR was performed with primers Hel/HuR5’ and Hel/HuR3\ which would yield a hinge region fragment of the Hel-N 1 cDNA coding region. To eliminate any DNA contamination, the samples were treated with DNase A. As the negative RT-PCR control (without MMLV-RT) shows, no DNA contamination was not observed. Because all the wildtype lung RNA were used in a Northern Blot experiment, only transgenic lung RNA was processed. As lane 1 and 2 in Fig 9 show, there was a 384 nucleotide PCR product representing the predicted PCR product of hinge region. The band was not seen without the presence of primer. The RT-PCR and Western Blot confirms that the transgene was transcribed to mRNA, which was then translated to Hel-Nl protein in lungs of the transgenic animals. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion After the first series of injections, two transgenic founders were obtained. At least two independent lines are required to ensure that it is the expression of protein, rather than the site of insertion that causes any observed phenotypes. Both founders appeared to be healthy and were both producing healthy litters. The transgene insertion did not interfere with their fecundity or their daily functions. Of the two founders, IA17 seemed to be carrying two only copies of the transgene, while IA18 seemed to carry many more. Both seemed chimeric based on band intensity on the Southern Blot when compared with the Southern Blot with IA 29 (IA17 progeny). The bands on the founder were much weaker than the transgenic progeny. However, only IA 17 has yielded germline transmission, while IA 18 has yet to produce transgenic progeny. It has been observed that sometimes high copy founders have a hard time at establishing germline transmission or expressing protein of interest, (personal communication from Dr. Rob Maxson of Norris Transgenic Core). The theory is that high number of copies inserted may result in the formation of heterochromatin insertion, and thereby turning off the gene. The mechanism for this is not proven, and is not under any active study. The DNA obtained for PCR and Southern blot was by tail biopsy method. In doing so, we were assuming that the tail DNA is an accurate representation of the number of inserted transgene for the rest of the body. In cases where the founder is chimeric, such assumption may be wrong. Our IA 18 founder, despite indication 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of high copy number from tail DNA, could have no transgene inserted in the germline cells, therefore was unable to produce any transgenic progeny. Using Western Blot analysis and RT-PCR, we were able to detect the presence of Hel-Nl cDNA and Hel-Nl protein in transgenic mouse lung. Preliminary experiments aimed at confirming the specificity of lung specific Hel-Nl expression are still being performed. Western Blot and RT-PCR will be performed with various tissue samples obtained from both wildtype and transgenic animals. It is interesting to note that even though both transgenic male and female pups have been produced, they came exclusively from female transgenic animal mating with a wildtype male. Thus far, the mating between a transgenic male and any female mouse, whether transgenic or wildtype, has not produced any litter. A total of seven transgenic males are currently being mated and none has produced a litter yet. It is not known whether overexpression of Hel-Nl could cause male sterility. If this condition persists, another transgenic line would have to be generated in order to produce a homozygous animal, as well as testing if Hel-Nl overexpression does cause male sterility in mice. Relying on data from previous experiments that the CC10 promoter would give lung specific expression, we have established a transgenic mouse line that has Hel- Nl expression in lung. Would the neuronal differentiation in lung be enough for the development of SCLC? We do not anticipate that SCLC would develop by simply pushing the lung toward a neuronal differentiation. Assuming that Hel-Nl would cause mRNA stabilization and myc gene activation, that would account for at least • 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. one possible step toward oncogenesis. Other cell cycle regulatory genes such as p53 or pRb must also be inactivated, for lung cancer to develop. In fact, p53 and pRb were found to be inactivated in the majority of SCLC ( ), thus Hel-Nl induction alone would not be sufficient to develop SCLC. Dr. Francesco DeMayo (Baylor) has a FvB/N transgenic strain that carries SV40 Large T Antigen (TAg) under the control of CC10 promoter. Tag has been shown to disrupt normal cell cycle control by binding and inhibiting the cell cycle checkpoint proteins pRb and p53. Dr. DeMayo’s transgenic mouse strain develop lung adenocarcinoma at the age of 4 months. (18) Our CC10- Hel-Nl strain will be crossed with Dr. DeMayo’s strain. In doing so, we would combine Hel-Nl expression, which may lead up to myc gene activation, and TAg expression which would inactivate p53 and Rb, all in one cell type. Similar experiments have been done by the lab of Dr. Ball (as yet unpublished results). A TAg mouse was crossed with a FvB/N mouse under the control of CC10 promoter, expressed hASH-1, a neuronal transcription factor protein also observed in 50% of SCLC. The cross yielded mice with adenocarcinoma, with small neuroendocrine foci, but no SCLC, although none were viable. Since Hel-Nl is also a neuronal differentiating protein, the cross between TAg mouse and our strain should develop lung cancer. We hope that Hel-Nl would be the neuronal differentiation needed for the cells to go down the SCLC pathway. If the TAg mouse cross with our strain does not provide the sufficient push for cancer development, then hASH-1 mouse can be crossed with our mouse first, then with TAg mouse. Dr. Ball’s hASH-1 mouse did not develop any 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cancer before it was crossed with TAg mouse. In summary, I have successfully developed a transgenic mouse strain which expresses neuronal protein, Hel-Nl, in lung. Another injection oftransgene will be required to generate at least another founder line (currently underway). Morphological changes of the lung of transgenic and wiltype mice are in progress of analysis. Based on the role of Clara cells in lung repair, PNE cell differentiation, and their position in the lung, as well as Dr. Ball’s preliminary data, we believe that our approach is sound. The deletion of chromosome 3p is almost always observed in SCLC patients, but its effect on the development of the disease is still a mystery. The identity of the deleted gene(s) is still unknown. If we are successful in developing an animal model for SCLC, it would be useful in trying to discern the molecular pathway that leads up to SCLC development, as well as for the possible development of new diagnostic and treatment procedure. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Fig. la Not I Site Knockout ofpKS-CCIO Plasmid T3ncuh XH3 pKSCCIO pKSCCIO G953 G954 marker plasmid x Not I x Not 1 x Not I « a f (Fig. la) Clones were digested W ith. Not I to see if the Nat I had been destroyed. Any sample that wasn't linearized could be a potential clone. G954 was chosen, and subsequent double digests were done to confirm that Not I site had been destroyed. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. X < n o > • c R ] 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. & Q U - 3 • § o O J w ■ D cn SJ ^ tn w O s 3 4 4 " ' O 5 rtf 6 £ /• j a, 5 ? n i--^ - * - 2 - s ; ^ &§ i— I « u 0 0 * 1 i- < W f T — I o la 4 - 4 S 3 a> 1 « — 1 w o } — I c < ^ P r ? & «$$$ M j > d J 4 1 III) - B - S S s f t . ■ * 1 " * r t ' £ S 3 C M O V 'lC O < ^ 3 C M Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Fig. 3 a Hel-Nl Gene Lisertioit pKS- CC10 23 25 26 27 28 29 30 31 32 33 34 x Bam HI marker (Fig. 3a) To confirm whether H el-Nl sequence was inserted, the DNA of the clones were digested with Bam HI. The samples that were linearised and running higher than the control are potential clones. H33 was later chosen and confirmed by sequencing with primer CClOSequencer. u > w 4 > ST 4 - S . - : a % s ’ H o ® s . ■ 5 * « & • *0 < * * 1 Si ■ f « i I ♦ t JR .2' Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (F ig 3 b ) H 3 3 w as digested w ith various enzymes u sed f o r isolating th e trangene t o make sure, that th e enzyme d id work. to v X pi 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Fig 4 U > o © PCR analysis of founder candidates (IA. 13 - 19) XH3 lkB 13 14 15 16 17 18 -Pr-D NA Plasmid Cont. 1 2 3 4 5 6 7 8 9 10 11 12 (Fig. 4) 3 males (IA 13-15) and 3 females (IA16-18) were analyzed by PCR tail DNA. Transgenic mouse should give rise to the same band as plasmid control (lane 12), as IA17 (faintly) and IA18 samples do Lane 10 and 11 are negative controls lacking either primer or DNA. Land 1 and 2 are molecular markers (XHiadl and IkR ladder respectively) Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Fig 5 A VO x BarM l x EcS.\ (A # 13141516 17 13 13 141516 1718 10- 8 - »' — — - - 8 5- 4._ * 1 2 3 4 5 6 7 8.9 1011 12 Southern blot analysis of founder (Fig 5) DNA samples were digested with either Bam HI or EcoR I, separated on a agarose gel, then transferred onto a Zetabind membrane. Using a Hel-Nl hinge region probe, the fragments around 3 kb are transgenic insertions (possibly arising from concatemericly inserted copies) The higher bands are either flanking copies or incomplete digests. Comparison of lane 11 and 12 using the phosphoimager indicates that there is around a 6 fold difference between the signal in the two lanes. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. x Brim HI x EcoR I IA 24 2526 27282930 24 25 26 27 28 29 30 A ■liB ■ 1 1 1 3 kb. - t * . o Noufiiein Blot of Ti .mgeiuc Progeny ;lsb f^Pigf g) IA29, a transgenic prog e rxv o f IA 17 lineage to show the difference in intensity of signals between -thecbi® eric founder and germline progeny. From the southern blot, one can see that IA23 has a signal equivalent to the endogenous bands on top, relative to IA17’s weaker signals. 4 > H is * *p«r s « s 4> W D 33 Sm S5 H ! ■ ■ ‘■ M * ■ v i ’ An- ,8 -|j < -g 5 mB ■ '< 1 1$ i tn Pw Ms • < U * # } '■ ■ ■ n f] V U. , r * m d = > : * 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Fig. S K > Transgenic fPos.t tNeg) W estern blot analysis of mice lung and brain, (Fig. 8) 5 p t,g (left of control lane) or 10 $ig (right of control) of each protein sample was loaded. The proteins were probed with human anti Hu protein serum. Pos. Lung lane has a faint signal band (arrow) while Neg. Lung lane doesn’t 5 jj.g loaded ___ A , , "A 10 jig loaded A.____ A ... N eff :.Weg.v.iiosv: :;Pos. i j ? Neg. N eg Pos Pos Lung Ei am LungBi am Lung Brain LiuigBi am Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. P l !£ v t) GAP DH primer MMLV-RT Hinge primers Hel-Nl primer "f" H- - '* 4 * — 4" + < -» - H33 i * 1 kb ladder 1.5 kb 1 kb 0.5 kb ~ <f <f RT-PCR of Ti ■m'sjjeiu' ]Mij e Lung RNA (Fig, 9) R .T-PC R was performed on total RNA from a trangenic anim al. The RNA was treated with Rnase I before RT-PCR because of slight DNA contamination (see dark arrow). After RT-PCR, PCR was performed to isolate the 384 nucleotide Hel-Nl specific product (see white arrows). Negative control was done without prim ers. u > 1.5 kb 1 kb ft.Skb Reference 1. ACS. Cancer Facts & Figures, 1996. American Cancer Society: Atlanta. 2. Adamson, I. Y. R. 1996. Development of lung structure, in Lung cancer: principles and practice. Pass, H.I., Mitchell, J. B., Johnson, D. H. & Turrisi, A. T. (eds). Lippincott-Raven Publishers: Philadelphia, pp 663-670. 3. Baetscher, M., Schmidt, E., Shimizu, A., et al. 1991. SV40 T antigen transforms calcitonin cells of the thyroid but not CGRP-containing neurons in transgeinc mice. Oncogene 6:1133-1138. 4. Bemasconi, N. L., Wormhoudt, T. A. M., Laird-Offringa, I. A. 2000. Post- transcriptional deregulation if myc genes in lung cancer cell lines. Am. J. Respir. Cell. Mol. Biol. In press. 5. Carbone, D. and Kratzke, R. 1996. Rbl and p53 Genes, in Lung cancer: principles and practice. Pass, H.I., Mitchell, J. B., Johnson, D. H. & Turrisi, A. T. (eds). Lippincott-Raven Publishers: Philadelphia, pp 107-121. 6. Chen, H., Thiagalingam, A., Chopra, H., et al. 1997. Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression. Proc. Natl. Acad. Sci. USA 94:5355-5360. 7. DeMayo, F.J., Finegold, M. J., Hansen, T. N., et al. 1991. Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice. Am. J. Physiol. 261:L70-L76. 8. Donehower, D., Harvey, M., Slagle, B., et al. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nture 356:215- 221. 9. Fan, X. C. and Steitz, J. A. 1998. Overexpression of HuR, nuclear-sytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. EMBO J. 17:3448-3460. 10. Hoyt Jr., R. F., Sorokin, S. P., McDowell, E. M., McNelly, N. A. 1993. Neuroepithelial bodies and growth of the airway epithelium in developing hamster lung. Anat. Record 236:15-22. 11. Jain, R. G., Andrews, L. G., McGowan, K. M., et al. 1997. Ectopic expression of Hel-N 1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes. Mol. Cell. Bio. 17:954-962. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12. Johnson, B. E. 1996. The role of myc, jun, fos oncogenes, in Lung cancer: principles and practice. Pass, HI., Mitchell, J. B., Johnson, D. H. & Turrisi, A. T. (eds). Lippincott-Raven Publishers: Philadelphia, pp 83-98. 13. Johnson, D. H. 1996. Chemotherapy of small cell lung cancer, in Lung cancer: principles and practice. Pass, H.I., Mitchell, J. B., Johnson, D. H. & Turrisi, A. T. (eds). Lippincott-Raven Publishers: Philadelphia, pp 825-836. 14. Laird, P . W., Zijderveld, A., Linders, K., et al. 1991. Simplified mammalian DNA isolation procedure. Nuc. Acids Res. 19:4293-4296. 15. Levine, T. D., Gao, F., King, P . H., et al. 1993. Hel-Nl: an autoimmune Rna- binding protein with specificity for 3’ uridylate-rich untranslated regions of growth factor mRNAs. Mol. Cell. Bio. 13:3494-3504. 16. Linnoila, R. I. 1982. Effects of diethylnitrosamine on lung neuroendocrine cells. Exp Lung Res. 3:225-236. 17. Ma, W . J., Cheng, S., Campbell, C., et al. 1996. Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J. Biol. Chem. 271:8144-8151. 18. Magdaleno, S., Wang, G„ Mireles, V., et al. 1997. Cyclin-dependant kinase inhibitor expression in pulmonary clara cells transformed with SV40 Large T antigen in transgenic mice. Cell Growth and Diff. 8:145-155. 19. Malkinson, A. M. and Belinsky, S. A. 1996. The use of animal models of lung cancer in preclinical studies, in Lung cancer: principles and practice. Pass, H.I., Mitchell, J. B., Johnson, D. H. & Turrisi, A. T. (eds). Lippincott-Raven Publishers: Philadelphia, pp 273-284. 20. Maniatis, T., Fritsch, E. F., Sambrook, J. Molecular Cloning. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1982. 21. Manley, G. T., smitt, P . S., Dalmau, J., Posner, J. B. 1995 Hu antigens: reactivity with Hu antibodies, tumor expression, and major immunogenic sites. Ann Neurol 38:102-110. 22. Sandmoller, A., Halter, R., Suske, G., et al. 1995. A transgenic mouse model for lung adenocarcinoma. Cell Growth and Diff. 6:97-103. 23. Speirs, V., Bienkowski, E., Wong, V., Cutz, E. 1993. Paracrine Effects of bombesin/gastrin-releasing peptide and other growth factors on pulmonary neuroendocrine cells in vitro. Anat. Record 236:53-61. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24. STOS in Lung Cancer, September 14-15,1999. http://www.webtie.org/ sots/sots.htm 25. Taketo, M., Schroeder, A., Mobraaten, L. et al. 1991. FVB/N: An inbred mouse strain preferable for transgenic analyses. Proc. Natl. Acad. Sci. USA 88:2065-2069. 26. Tora, M., Graus, F., de Bolos, C., et al. 1997. Cell surface expresion of paraneoplastic encephalomyelitis/sensory neuronopathy-associated Hu antigens in small-cell lung cancers and neuroblastomas. Neurology. 48:735-741. 27. Wilkenheiser, K. A., Clark, J. C. C., Linnoila, R. I., et al. 1992. Simian Virus 40 Large T Antigen Directed by Transcriptional Elements of the Human Surfactant Protein C Gene Produces Pulmonary Adenocarcinomas in Transgenic Mice. Cancer Res. 52:5342-5352. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NOTE TO USERS This reproduction is the best copy available. __ ® UMI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UM1 Number: 1406440 ® UMI UMI Microform 1406440 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. 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Asset Metadata

Creator Chen, Taian (author)

Core Title A transgenic mouse model for SCLC: Expression of Hel-N1 in mouse lung

School Graduate School

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 2001-05

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Lee, Stuart (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-36892

Unique identifier UC11337191

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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