Molecular Biology C Conception, theory, research, and application ——Logic and LIY (Learn It Yourself) Sheng Zhao (赵晟), Biochemistry and Molecular Department of Medical school in Southeast University Couse QQ Club: 112342994 (分子生物学C ) Web: http://teaching.ewindup.info/ Email: [email protected] or [email protected] QQ /MSN/Skype/gChat: [email protected] Mobile:18551669724 or 13675130010 Chapter 4: Finding Scientific Nemo (From clinic samples to scientific research) Section 1: Trust it or trash it? ——Number, selection, and target (Establish sample library) Section 2: A colorful microscopic world ——Specific labeling (Visualization techniques) Case 4: Immortal clinic samples ——Primary culture and gene manipulation (Construction of the cell line) HeLa ---- An immortal cell line 吕一正 A HeLa cell, also Hela or hela cell, is a cell type in an immortal cell line used in scientific research. It is the oldest and most commonly used human cell line. Henrietta Lacks (August 1, 1920 – October 4, 1951) On January 29, 1951, she felt a knot inside her. She had told her cousins about the knot; they assumed correctly that she was pregnant. But, after giving birth to her fifth child, Joseph, Henrietta started bleeding abnormally and profusely. Her local doctor tested her for syphilis, which came back negative, and referred her to Johns Hopkins. Howard Jones, her new doctor, examined Henrietta and the lump in her cervix. He cut off a small part of the tumor and sent it to the pathology lab. Soon after, Lacks learned she had a malignant epidermoid carcinoma of the cervix. Two samples of Henrietta's cervix were removed—a healthy part and a cancerous part—without her permission.The cells from her cervix were given to Dr. George Otto Gey. Multiphoton fluorescence image of cultured HeLa cells with a fluorescent protein targeted to the Golgi apparatus (orange), microtubules (green) and counterstained for DNA (cyan). Multiphoton fluorescence image of HeLa cells stained with the actin binding toxin phalloidin (red), microtubules (cyan) and cell nuclei (blue). Scanning electron micrograph of an apoptotic HeLa cell. HeLa cells dividing under electron microscopy Scanning electron micrograph of just-divided HeLa cells. Chromosome number • Horizontal gene transfer from human papillomavirus 18 (HPV18) to human cervical cells created the HeLa genome which is different from Henrietta Lacks' genome in various ways, including its number of chromosomes. HeLa cells are rapidly dividing cancer cells, and the number of chromosomes varied during cancer formation and cell culture. The current estimate (excluding very tiny fragments) is a "hypertriploid chromosome number (3n+)" which means 76 to 80 total chromosomes (rather than the normal diploid number of 46) with 22-25 clonally abnormal chromosomes, known as HeLa signature chromosomes.The signature chromosomes can be derived from multiple original chromosomes making challenging summary counts based on original numbering. Researchers have also noted how stable these aberrant karyotypes can be. Telomerase • The HeLa cell line was derived for use in cancer research. These cells proliferate abnormally rapidly, even compared to other cancer cells. Like many other cancer cells, HeLa cells have an active version of telomerase during cell division, which prevents the incremental shortening of telomeres that is implicated in aging and eventual cell death. In this way the cells circumvent the Hayflick Limit, which is the limited number of cell divisions that most normal cells can later undergo before becoming senescent. Contamination • The degree of HeLa cell contamination among other cell types is unknown because few researchers test the identity or purity of alreadyestablished cell lines. • It has been demonstrated that a substantial fraction of in vitro cell lines — estimates range from 10% to 20% — are contaminated with HeLa cells. Stanley Gartler in 1967 and Walter Nelson-Rees in 1975 were the first to publish on the contamination of various cell lines by HeLa. In vivo magnetic resonance imaging tracking of bone marrow-derived mesenchymal stem cells via intracoronary administration: Consistency to pathohistological results 李浩 133772 Recent trials and clinical studies have shown that intracoronary transplantation of bone marrow-derived mesenchymal stem cells (MSCs) improves cardiac function following acute myocardial infarction (AMI). However, whether homing of MSCs into the infarcted myocardium or not is still unknown. Epicardial myocardium injection Endocardial myocardium injection Intracoronary injection Intravenous injection Methods Methods Myocardial infarction was induced in all 10 pigs Methods Making cell lines and gene deliver system Oncogenes including myc, bcl-xl, neu, p53, Ela, SV40, among which mcy is the most used. Deliver system: 1. Physical method: Electroporation, Nano particles 2. Chemical system: Bipolar molecules 3. Biologic system: Virus: wide spectrum, smaller inserts 1. Retroviral (genomic integration) Retrovirus (RV, replicative, ~10 kb genome) Lentivirus (LV, both replicative and non-replicative, ~10 kb genome) 2. Adenovirus (AD, epichromosomal, both replicative and non-replicative, ~10 kb genome) 3. Adeno-associated virus (AAV, 4.3 kb genome) Bacteria: Larger inserts, limited hosts cells 1. Salmonella sp 2. Clostridium sp 3. Escherichia coli comparing and optimizing approaches 133763 133764 蔡蓉蓉 胡姗姗 Objectives Investigate the feasibility of achieving high transgene expression in cultured muscle cells. Methods materials mouse C2C12 cells a novel system, primary rainbow trout myosatellite cells Methods three different viral vectors adeno-associated virus baculovirus (BAC) serotype 2 (AAV2) green fluorescent protein (GFP) lipofectamine 2000 lentivirus the genetic manipulation of cultured muscle cells 1. chemical/lipid-based transfection: Chemical transfections are fast and cost effective in most cell culture systems, but produce low transfection efficiencies in muscle cells. 2.viral vectors: capable of introducing transgenes in a tissue-specific manner and may or may not integrate transgenes into the host genome, depending on the specific vector The poor transfection efficiency of lipid-based protocols • GFP expression was lost as cells proliferated and was poorly retained after differentiation. • The reagent was toxic at very low cell densities. • In general, transfection efficiencies with cationic lipids are inversely related to transgene size. Adeno-associated viral vectors Adeno-associated viral vectors have great promise for gene therapy as they infect both dividing and nondividing cells, they rarely integrate into host genomes and they are relatively immune tolerant. Serotype:AAV2、AAV6、AAV8 AAV2 •In this study, we used AAV2 due to the comparatively lower transduction efficiency of AAV6 in vitro •In fact, a preliminary trial in our laboratory using both vector systems clearly demonstrated AAV2 superiority, even when vector was maintained on cells for 6 day •Disadvantage：The transgene expression did not continue after cells were passed BAC •BAC can transduce a broad range of mammalian cells as well and has emerged as a promising gene delivery vector due to low cytotoxicity and the ability to carry large inserts •It also transduced myoblasts and in a shorter time than AAV2, although a 10-fold higher titer was needed to produce the same relative transduction efficiency. Promoters CMV :The cmv activity is more variable in different cell types in vitro PGK: The pgk promoter was most effective in C2C12 cells although cmv and pgk appeared equally efficient in trout myosatellites, promoter activity of both was comparatively weak and could explain the need to use a 10-fold higher MOI to detect significant fluorescence. Lentiviral Lentiviral superiority over the other vectors is likely due to the fact that their genomes, the vector provirus, are integrated into host genomes. Our results also demonstrate that lentivirus itself does not alter the differentiation potential of C2C12 myoblasts. lentivirus effectively and rapidly infects myoblasts, at lower virus titers, and that transgene expression is maintained well after differentiation Can the nervous system regenerate ? The amount of neurons remains unchanged and the nervous system loses the ability of proliferation and differentiation since one person is born? 何骏驰 • The immortalization of wild type hNSC with v-myc results in the establishment of a stable neural stem cell line (v-IhNSC) with the ability to originate mature functional neurons and conspicuous amounts of oligodendrocytes in vitro. • 1. Propagation and differentiation of neural stem cells • Cells were derived from brain of a aborted human fetus, cultured in the serum-free medium containing all needed nutrition, and were induced to differentiation. • 2. Retroviral infections • Retroviral vector encoding v-myc was used to transduce hNSCs at 17 passages from the primary culture. A mock vector was used as a control. Retroviral transduction was a repeated infection procedure. Then v-IhNSCs were induced to differentiation.