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BIOL483- notes starting after pg 36 ie after midterm.docx

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BIOL 483
Vivian Dayeh

CELL LINE AND CULTURE MONITORING - Cell cultures are to be monitored to assess the condition of the culture - Cells can be counted manually or electronically CELL COUNTING AND MONITORING Hemocytometer - Manual cell counting technique - Consists of thick glass slide with a grid - Cells are counted and the total number of cells/ml can be determined Coulter Counter - Electronic cell counting technique - Is a much faster method - Solution is drawn up through a tube, creating a pulse that is detected by the counter - We can set an upper and lower size limit of particles to avoid particles that aren’t the size of the cells that you want to count INDIRECT METHODS FOR CELL MONITORING - Protein Determination o Measures the total amount of protein o Protein levels in cells can vary o Bradford assay is used to determine the protein content - Glucose Determination o Measures the changes in glucose levels with respect to cell density o This technique is a good choice for immobilized cells, when it is hard to isolate cell samples in bioreactors, due to immobilization CELL VIABILITY - Cell viability measures the metabolic state of a cell population - In other words, cell viability is the potential for cell growth in culture - We can measure cell viability to determine the proportion of cells that are live and metabolically active CELL VIABILITY TESTS: - Colony Formation o This is a direct measure of the ability of cells to grow o Plate a known number of cells into a petri dish, and each viable cell will grow and produce a distinct colony Cell Membrane Integrity (being whole, undivided) 2 types of assays - Dye Exclusion Assay o Dye used is TRYPAN BLUE o The dye will penetrate DEAD cells and stained blue o The number of stained cells to non-stained cells will indicate the viability of the culture - Retention of a marker molecule o This is measuring the ability of cells to retain the marker molecule o LDH is a common enzyme used o Dead cells will release LDH into the medium, and we will measure the LDH activity o Other markers are CFDA (carboxyfluorescein diacetate) and CF (carboxyfluorescein) o CFDA  non-polar and non-fluorescent dye o CF  polar and fluorescent Metabolic Activity  2 examples on measuring metabolic activity 1) Tetrazolium Assay - Measuring Mitochondrial Activity - Uses the MTT reagent, which is yellow - Via spectrophotometry, viable cells will turn blue - Viable cells are active 2) Resazurin Reduction Assay - Measuring Metabolic Activity - Uses a non-fluorescent dye that will permeate the cell membrane - Dye will go from dark blue to purple/pink GENETIC MODIFICATION We are now capable of genetically manipulating cells to express proteins of our choice In order to genetically modify cells in culture you need four things… 1) Gene of Interest – the sequence we want to introduce into the cells 2) Cells- can choose to transfect either a WT or Mutant cell 3) Transfection Mechanism- either a natural or artificial way to introduce the GOI 4) Selection Mechanism- a dominant or selectable marker, to determine if uptake of GOI has been successful HOW TO GET DNA INTO MAMMALIAN CELLS.. Getting cells to fuse, we can use FUSOGENS There are 3 types of fusogens o Biological o Chemical o Electrofusion 1) Fusogens cause proteins in the membrane to move around - This will create a region in the cell without any proteins, these areas are unstable and allow for cell fusion - There are 4 cell fusion products o Homokaryon o Heterokaryon o Synkaryon o Cell hybrid 2) CHROMOSOME TRANSFER - We can transfer chromosomes via several techniques Isolated chromosomes Arrest chromosomes at metaphase Microcells  arrest cells in mitosis, will form several small nuclei, centrifuge and use fusogens to introduce the microcell into another cell 3) Transfection - Is a direct method for direct transfer of nucleic acids into animal cells o Extract DNA o Cut with RENS o Incorporate into plasmid with selectable markers o Clone in bacteria o Transfect into cell line o Use selectable media to grow transfected cells How do we introduce the DNA into the cells, to express the gene of interest?  Via artificial or natural transfection Artificial Transfection - Direct Methods o Microinjection, injection is not specific, would hope DNA uptakes material - Indirect Methods o Calcium Phosphate Method o DEAE Dextran o Liposomes o Protoplast fusion o Electroporation o Microprojectiles Natural Transfection Uses Viruses to manipulate the cells in culture - A very efficient way to introduce genes is via TRANSDUCTION when using a viral vector SV40 Virus- simian virus - Causes neoplastic transformation in vitro - There are 2 phases of transcription in SV40 infection o Early phase- t antigen proteins made to unwind viral DNA o Late phase- transcription and production of capsid proteins - This will eventually lead to cell lysis and release of SV40 - The virus will infect the cell and introduce its foreign DNA - ONLY DISADVANTAGE is that you require a permissive cell line derived from a monkey RETROVIRUSES - These are RNA viruses and can incorporate genes into nuclear DNA - This requires cDNA sequences from RNA Regardless of transfection method, only a tiny proportion of cells will actually take up and express the DNA that you introduce - The selectable gene that is used depends on whether WT cells or mutant cells are used We can use Nucleotide Biosynthesis as markers for transfection of GOI, this can determine state of transfection (ie
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