CYTBT-I 2013-4.pdf
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Conceptos y Técnicas de Biotecnología I 2013-2ºC FBMC-FCEN-UBA Transferencia de Genes a Células Animales en Cultivo I Unidad de Transferencia Genética Instituto de Oncología “Ángel H. Roffo” Universidad de Buenos Aires ORGANISMOS TRANSGÉNICOS - INVOLUCRADOS EN BIOTECNOLOGÍA •BACTERIAS (i.e.: Escherichia coli) •HONGOS (i.e.: Saccharomyces cereviseae) •CULTIVOS CELULARES (animales o vegetales) •PLANTAS ALGAS VASCULARES •ANIMALES: PECES AVES MAMÍFEROS Bovinos Caprinos Ovinos Porcinos HUMANOS: Transgénesis parcial Terapia Génica Transferencia de Genes a Células Animales en Cultivo PROPÓSITOS •Confirmar identidad de genes •Caracterizar oncogenes •Expresar proteínas que necesitan modificaciones posttraduccionales •Producir grandes cantidades de proteínas que naturalmente se encuentran en cantidades limitadas •Estudiar síntesis y transporte intracelular •Expresar secuencias genómicas que contienen intrones •Estudiar mecanismos de edición de genes •Analizar señales de control de transcripción y su modulación por drogas, hormonas, estado de diferenciación Heterologous Protein Production In Eukaryotic Cells • Prokaryotic systems are generally cheaper, but… • Eukaryotic proteins produced in bacteria may be Unstable or lack biological activity due to lack of posttranslational modifications or correct assembly Possess unacceptable contaminants after purification Posttranslational Modifications • • • • • Conformation Cleavage Correct disulfide bond formation Protein disulfide isomerase Amino acid removal from initial polypeptide • O-linked or N-linked glycosylation About 30% of eukaryotic proteins are glycosylated Examples of O-Glycosylations Examples of N-Glycosylations Examples of N-Glycosylations • Initial groups can be trimmed and then expanded to create different final modifications Examples of N-Glycosylations • Another Variation Cultivate mammalian cells!!!! Carrel (surgeon, 1923) Aseptic techniques Carrel Flask 50-s Chemically defined media (Eagle, Earle) Consistency Sterilization Reduced chance of contamination Growth of Animal Cells in Culture • In vitro cell culture systems enable scientists to: – study cell growth and differentiation – perform genetic manipulations to understand gene structure and function. • Culture media contains: – Serum – Salts – Glucose – Various amino acids and vitamins that the cells do not make for themselves. Ejemplo de medio de cultivo de células de mamífero: Dulbecco Modified Eagle medium (DMEM) Vitamins Inorganic Salts Calcium Chloride 0.2 0.2 Choline Chloride 0.004 0.004 Ferric Nitrate • 9H2O 0.0001 0.0001 Folic Acid 0.004 0.004 Magnesium Sulfate (anhydrous) 0.09767 0.09767 myo-Inositol 0.0072 0.0072 Potassium Chloride 0.4 0.4 Niacinamide 0.004 0.004 Sodium Bicarbonate 3.7 3.7 0.004 0.004 Sodium Chloride 6.4 6.4 D-Pantothenic Acid (hemicalcium) Sodium Phosphate Monobasic (anhydrous) 0.109 0.109 Pyridoxal • HCl — — Pyridoxine • HCl 0.004 0.004 Riboflavin 0.0004 0.0004 Thiamine • HCl 0.004 0.004 D-Glucose 4.5 4.5 Phenol Red • Na 0.0159 — Pyruvic Acid • Na 0.11 — 0.584 0.584 Amino Acids L-Arginine • HCl 0.084 0.084 L-Cystine • 2HCl — 0.0626 Glycine 0.03 0.03 L-Histidine • HCl • H2O 0.042 0.042 L-Isoleucine 0.105 0.105 L-Leucine 0.105 0.105 L-Lysine • HCl 1.46 0.146 L-Methionine — 0.03 L-Phenylalanine 0.066 0.066 L-Serine 0.042 0.042 L-Threonine 0.095 0.095 L-Tryptophan 0.016 0.016 L-Tyrosine • 2Na •2H2O 0.10379 0.6351 L-Valine 0.094 0.094 Other Add L-Glutamine + compuestos no-definidos: suero sanguíneo (5-15%), cocktail de factores de crecimiento, etc. Serum • 0-20% Serum – – – – – – Growth factors Transferrin (Fe) Lipids Insulin Shear protection Detoxification Problems – Infectious agents (viruses, mycoplasm, prions) – serum composition is poorly defined and the batches vary. – Expensive Completely mammalian origin free (MOF) chemically defined medias Growth of Animal Cells in Culture • Primary cultures are the original cultures established from a tissue. • Permanent (or immortal) cell lines are embryonic stem cells or tumor cells that proliferate indefinitely in culture. Growth curves in yeasts and mammalian cells are different 10.0 Stationary Cx (g/l) 1.0 decline 0.1 exponential Yeast Hybridoma 0.0 0.0 Minimum 0.0 density Lag phase 0.0 0 50 Time (h) 100 Ejemplo: historia del desarrollo de la línea HEK-293 Graham, et al., J. Gen. Virol., 36:59-72, 1977 Dificultades suplementarias • El tiempo de división aumenta con la talla: – Las células animales necesitan condiciones de asepsia muy estrictas • Adhesión obligatoria: – Ciertas líneas de células de mamíferos necesitan un soporte para ser viables. Esto: – presenta un problema de escalado – las vuelve particularmente susceptibles a la disrupción Ej: tapiz celular de mioblastos Cultivo sobre microcarriers MODALIDAD •Transitoria: s/ Integración: Expresión 12-72 horas •Permanente: c/ Integración: Expresión 1-3 semanas PARÁMETROS A CONSIDERAR •Tipos celulares disponibles •Expresión transitoria o permanente (estable) •Elementos de control de expresión adecuados Common cell lines CHO Epithelial Chinese Hamster Ovary HeLa Epithelial Human cervical carcinoma MDCK Epithelial Canine Kidney BHK Fibroblast Baby Hamster Kidney Vero Fibroblast Monkey Kidney WI-38 Fibroblast Human fetal lung 3T3 Fibroblast Mouse fibroblast MARCADORES FENOTÍPICOS •Crecimiento en agar •Crecimiento con bajo suero •Cambios morfológicos •Rescate de la muerte de cultivos celulares primarios MARCADORES BIOQUÍMICOS Indicadores •Anticuerpos específicos •Chloramphenicol acetyltransferase (cat) •β-galactosidase (β-gal) •Luciferase Selectores •Thymidine kinase (tk) •Xantine guanine phosphoribosyl transferase (xgprt) •Aminoglicoside phosphotransferase (apht/neor) •Dihydrofolate reductase (dhfr) •Hygromycin B phosphotransferase (hygr) Indicadores/Selectores •Green/Blue/Red fluorescence proteins (gfp/bfp/rfp) MARCADORES BIOQUÍMICOS Indicadores •Anticuerpos específicos Ag + AbI Ag.AbI Ag.AbI + AbII. Ag.Ab.AbII. : enzima, grupo fluorescente, grupo radioactivo •Chloramphenicol acetyltransferase (cat) [14C] Chloramphenicol Ac- [14C] Chloramphenicol (Ac)2-[14C] Chloramphenicol •β-galactosidase (β-gal, lac Z) lactosa galactosa + glucosa ONPG o-nitrofenol (amarillo) + galactosa Xgal X (azul) + galactosa •Luciferase Luciferina + ATP Luciferina + ADP + hν (luz) Indicadores/Selectores •Green/Blue/Red fluorescence proteins (gfp/bfp/rfp) XFP + hν1 (luz) XFP + hν2 (luz) ν1 >ν ν2 Selector: FACS Fluorescence-activated cell sorter Reporter gene systems 1. chloramphenicol acetyl transferase (CAT) CAT is a bacterial enzyme that catalyzes the transfer of acetyl groups from acetyl-coenzyme A to the antibiotic chloramphenicol. (chloramphenicol deactivation) thin-layer chromatographic sheet Chloramphenicol is radiolabelled For mammalian cells it is laborous and expensive. Extract protein and measure activity… β-galactosidase (βgal) systems luciferase (luc) systems firefly species Photinus pyralis Expressed luciferase catalyses oxidation of compounds called luciferans ( ATP-dependent process) mouse with a strain of salmonella luciferans emit fluorescense luminometer measurement Mice are injected with LUC+ salmonellas. Sensitive digital cameras allow non-invasive detection. For GT vectors pictures look the same Green fluorescent protein (GFP) autofluorescent protein from Pacific Northwest jellyfish Aequorea victoria GFP is an extremely stable protein of 238 amino acids with unique post-translationally created and covalently-attached chromophore from oxidised residues 65-67, Ser-Tyr-Gly ultraviolet light causes GFP to autofluoresce In a bright green color Jellyfish do nothing with UV, The activate GFP by aequorin (Ca++ activated, biolumuniscent helper) Green fluorescent protein (GFP) GFP expression is harmless for cells and animals GFP transgenic mice from Osaka University (Masaru Okabe) GFP construct could be used for construct tracking in living organism GFP labelled image of a human tumor. Vessel on the tumor surface are visible in black MARCADORES BIOQUÍMICOS Selectores •Thymidine kinase (tk):) dT +ATP dTMP +ADP dUMP dTTP ½ select. céls. tk-: HAT (hipoxantina, aminopterina, timidina) inhib: aminopterina •Xantine-guanine phosphorybosil transferase (xgprt) Xantina →XMP → GMP ← ← guanina ↑ inhib: ac. aminofenólico precursores → → IMP inhib: aminpoterina ↓ ASMP → AMP ½ selectivo p/toda cél.: AAMX (adenosina, aminopterina, ac. Aminofenólico, xantina) ASMP: ac. adenilosuccínico •Aminoglicoside phosphotransferase (apht/neor) neomicina/kanamicina/geneticina (G418) → antibiótico fosforilado (inactivo) •Dihydrofolate reductase (dhfr) DHF→ →THF ½ selectivo en céls. dhfr-: ausencia de nucleósidos ½ selectivo p/toda cél.: methotrexate (MTX) •Hygromycin B phosphotransferase (hygr) Hygromicina B → antibiótico fosforilado (inactivo) Gene amplification
