4Recombinant DNA Technology in the Synthesis of Human Insulin
5DIABETES and the role of Insulin Diabetes mellitusGreek for “siphon” and Latin for “Honey”Characterized by excretion of large amounts of sugar in the urineResults from the body’s inability to make sufficient insulin, hormone involved in the regulation of blood sugar (glucose)Insulin is secreted into the bloodstream from pancreatic cells where it signals the appropriate tissues (liver and muscle) to remove the excess glucose from the blood.
6Types of Diabetes Insulin-dependent or Juvenile-onset Caused by lack of insulinAffects mostly childrenNon-insulin-dependent or adult-onsetDue to deficient insulin receptorsMalfunctioning communication system within the body
7Regardless of diabetes type, same principle works Insulin recognizes specific insulin receptors on particular cells and initiates a cascade of reactions that results in the uptake of glucose.Regardless of diabetes type, same principle worksDespite high levels of glucose in the bloodstream, the proper signal does not trigger its uptakeIndividual cells begin to starve even though plenty of glucose is available.
9What happens in the absence of Insulin ? No glucose uptakeCells begin to use fats as primary source of energy.Catabolism of fats results in synthesis of ketone bodies,Ketone bodies (acetone) are secreted into the bloodstream and function as alternative source of energy for the brain (cannot utilize fat directly)Excess of ketone bodies are harmfulBlood becomes acidicToxic at high levelsExcretion of glucose and ketones in the urine carrying along huge amounts of water and salts, severe dehydration
10Reaction of muscle cells Muscle cells, requiring large amount of glucose for ATP synthesis, react the starvation by metabolizing proteinsLarge amount of ammonia is produced (toxic to human)Normally converted into to urea and excretedAmmonia can rise to toxic level under diabetic conditions
11A simple breakdown in communication results in greatly altered metabolism in many cells. The long-term effect of these changes can include kidney failure, heart disease, brain damage, and ultimately death.
12Treatment of Diabetes Non-insulin-dependent Insulin-dependent Through dietWeight reductionInsulin-dependentUsing insulin (serves to bring insulin levels to normal)Requires ready supply of insulin
13Insulin Production Earlier, extracted from pancreas of cows and pigs Organ (pancreas) was obtained from slaughterhouses for insulin extractionDrawbacksDue to increasing incidence of insulin-dependent Diabetes, an increased supply of insulin was requiredAvailability of pancreas decreased due to decreased consumption of red meatAlternative source of insulin desirable
14Genetic engineering for Insulin production Creation of genetically engineered bacterial cells that produce human insulinGeneration of “Bacterial Factories” that can produce cheap, readily available source of insulinPotential benefits of recombinant insulinReady source of product (bacteria easy to grow)No allergic reaction to animal insulin
15General considerations Biological reactions inside and outside the cellsIsolation and purification of functional polymerasesNo interference or contamination from other biological moleculesGrowing bacteria in the laboratoryNeed to grow bacterial cells without contamination in specific medium according to the requirementBroth (to get large quantities of cells)Agar (colony formation, to get pure culture and to observe unique properties)
17in genetic engineering 3. Detecting what happened to individual moleculesCan we see DNA?Gene?How will we know that the tube contains DNA?How can we say that we are manipulating out desired piece of DNA?All these issues are of primary importancein genetic engineering
18Cloning and expression of Insulin Obtain the gene for insulin from human DNAInsert the gene into bacterial cellsSelect cells that have desired geneInduce the bacterial cells to express “foreign” gene in order to produce insulinCollect and purify the final product, insulin
191. Obtaining the insulin gene Find the piece of DNA that codes for insulin among the rest of the DNA that makes up human geneHow?The most common methodIsolate mRNA rather than DNAMore copies of mRNA than the coding gene itselfIf obtained from pancreas, very high copy numberHave poly A tail on 3’ end (help in isolation of mRNA)
20Conversion into cDNA and amplification of gene Reverse Transcription using reverse transcriptase (RT)Synthesizes complementary strand of DNA using template mRNA (cDNA)DNA polymerization using DNA polymerasePolymerase chain rection
24PCR Requirements DNA (purified or a crude extract) Primers specific for the target DNAFree nucleotides (A, G, T, C)DNA polymeraseBuffer (containing magnesium)
25PCR Primers Usually about 18-26 nucleotides in length Designed to flank the region to be amplifiedGC content between oCMelting point determined by G-C and A-T contentTm = 4oC (G+C) + 2oC (A+T)Ex: a primer with 10 G/C and 10 A/T would have a Tm of 60 oC (10) + 2(10)= 60 oCTm of both primers within 2 oCAvoid hairpin, dimer and self dimer
262. Inserting genes into bacterial cells Can we insert a piece of DNA (PCR amplified) into cell?Linear DNA does not enter the cell easilyBacterial cells do not tolerate DNA that does not form circular structures, linear pieces are destroyedIt will not contain the proper signals of transcription, translation and replication systems.
27Use of vector for gene insertion Genes must be incorporated into vectors (carriers) for safe introduction into bacterial cellsVectors are moved between test tube and the cellMost common vectors are PlasmidsCircular pieces of DNA found in different micro- organisms and are replicated independent of the chromosomal DNAUsually contains few genes, sometime only one (antibiotic resistance gene)
28Basic Properties of Plasmids Small, easily manipulated DNA moleculesEncode genes for antibiotic resistanceCan be readily transferred into cells and can be isolated easilyPlasmid contains signals for independent replication within cells.Contains multiple but unique cloning sitesDNA inserted into plasmid will be replicatedalong with the plasmid DNA
30Tools of the trade Restriction endonucleases (molecular scissors) DNA LigaseRibonucleasesTerminal transferasePolynucleotide kinaseAlkaline phosphatase
31Restriction endonucleases Enzymes that attack and digest internal regions of the DNA of an invading bacteriophage but not that of the host.First enzyme extracted from E. coli (cut randomly and not always close to the desired site).They break the phosphodiester bonds that link adjacent nucleotides in DNA molecules.Cut (hydrolyse) DNA into defined and REPRODUCIBLE fragmentsCleave DNA in a sequence-specific mannerMost restriction enzymes cut DNA which contains their recognition sequence, no matter what the source of the DNA is.Evolved as a defense mechanism against infection by foreign DNADifferent restriction enzymes in different organisms
33Type II RE 3 types of cuts - 5’ overhang, 3’ overhang, blunt 5’-GAATTC-3’3’-CTTAAG-5’5’-GAATTC-3’3’-CTTAAG-5’2) 3’ overhang5’-CCCGGG-3’3’-GGGCCC-5’3) blunt
34REs as bacterial defense system In the bacterial strain EcoR1, the sequence GAATTC will be methylated at the internal adenine base (by the EcoR1 methylase).The EcoR1 endonuclease within the same bacteria will not cleave the methylated DNA.
35Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes
36How will you proceed for insertion of gene into vector ? No restriction site on the ends of PCR amplified geneIf there is restriction site ?If the sticky ends are compatible ?If the ends are incompatible.One end is sticky and the other is bluntBoth ends are sticky but incompatible