Molecular Biotechnology MCB 4312
University of Central Florida
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Date Created: 10/22/15
1 Immunological Diagnostic Procedures a ELISA i Direct ELISA 1 No secondaw Ab necessaw ii Indirect ELISA 1 Bind the sample being tested for the presence of a speci c molecule or organism to a solid support such as a plastic microtiter plate which usually contains 96 sample wells Wash the support to remove unbound molecules 2 Add a markerspeci c Ab primaw Ab directed against the target antigen to the bound material and then wash the support to remove unbound rima 3 Add a second Ab secondaw Ab that binds speci cally to the primary Ab and not to the target molecule a Bound conjugated to the secondary Ab is an enzyme that can catalyze a reaction that converts a colorless substrate into a colored product e mixture to remove any unbound secondaw Abenzyme conjugate 4 Add the colorless substrate Obsene or measure the amount of colored product iii Sandwich ELISA Plate is coated with a capture Ab 2 Sample is added and any Ag present binds to capture Ab 3 Detecting Ab is added and binds to Ag 4 Enzymelinked secondaw Ab is added and binds to detecting Ab 5 Substrate is added and is converted by enzyme to detectable form iv Variations 1 Abs attached to well a Patient sample added containing Ag Ag will bind to the Ab in the well b Peroxidaseconjugated antibody added c To identify the presence of target protein in a sample 2 Serology a Bacterial Ag af xed to substrate Patient serum added b If Ab v Ag present it will stay in the well c Peroxidaseconjugated antihuman IgG Ab added color reaction antibody in serum recognizes Ag Testing for Ab production in response to antigen 3 Seronegative No speci c antibodies to a pathogen no prior exposure 4 Seropositive a Production of speci c antibodies to a pathogen 5 Seroconversion a Switch from seronegative to seropositive 2 Monoclonal Antibodies a Monoclonal antibodies mAb or moAb are monospeci c antibodies that are identical because they are produced by one type of immune cell that are all clones of a single paren cel b Each Ab binds only one speci c antigen c Types i Murine monoclonal Abs Chimeric and humanized monoclonal Abs Human monoclonal A s 1 Produced by merging the DNA that encodes the binding portion of the monoclonal mouse Ab with uman Abproducin DNA d HAT Procedure Hypoxanthine Aminopterin Thymidine 39 s Immunize Mouse gt Isolate Spleen cells Myeloma Cells HGPRT Nonfused Spleen Cells S Nonfused Myeloma Cells M Fused SpleenSpleen cells SS Fused MyelomaMyeloma Cells MM Fused Hybrid SpleenMyeloma Cells SMgt 4 Addition of HAT Mediumgt a M and MM cannot grow S and SS cannot divide OR b SM grow and divide 39 Sunive in HAT because the spleen cell contributes a functional HGPRT which can utilize the exogenous Hypoxanthine in the medium even though purine production by means of dihydrofolate reductase is blocked by aminopterin and because the cell division functions of the myeloma cell are active e Production and Screening of Mabs 39 Spleen cells from a mouse that was immunized with a speci c antigen are isolated and fused in culture with myeloma cells that do not produce antibody chains ii Fused cells are selected for the ability to grow on HAT medium which contains previously mentioned 5 WWI 1 iii Cells that produce a speci c antibody the immunizing Ag hybridomas are identi ed by an immunoassay and individually subculture iv A hybridoma which grows in culture and secrets a single type of antibody molecules is the source of a monoclonal antibody f Targets for Diagnostic Mabs i Polypeptide Hormones Chorionic gonadotropin 2 Growth 0 3 Luteinizing hormone 4 Folliclestimulating hormone 5 Thyroidstimulating hormone 6 Prolactin mor Markers Carcinoembryonic antigen 2 Prostatespeci c antigen 3 Interleukin2 rece tor 4 Epidermal growth factor receptor iii Cytokines 1 Interleukins 18 Colonystimulating factor iv Drug Monitoring Theophylline 2 Gentamicin 3 Cyclosporin v Miscellaneous Targets oxine Vitamin B12 Ferritin Fibrin Degradation Products Tau protein ctious Diseases Chlamyd39a ii Tu vi 5 mwwr3wwNH He B Legionella HIV 3 Colored Fluorescence Proteins i 238aa long photoprotein Isolated from jelly sh Aequorea Victoria 0 n i Isolated from Discosoma coral ii Production of monomeric red uorescent protein requires 33 mutations c Luciferase i Come from U genes re y uc genes Includes ve genes uXCDABE iii Peaks at 490nm iv Genes A and B are responsible for generating the light signal 4 DNA Diagnostic Systems General Laboratow Nucleic Acid Hybridization Scheme i Bind ssDNA target to a membrane support ii Add singlestranded labeled DNA probe under appropriate conditions of temperature and ionic strength to promote base paiting between the probe and the target DN s iii Wash the support to remove excess unbound labeled probe DNA iv Detect the hybrid sequences that form between the probe and target DNA b Chemiluminescence i Biotinlabeled probe is bound to the target DNA ii Streptavidin is bound to the biotin molecules iii Biotinlabeled AP binds to Streptavidin iv AP converts the substrate into a lightemitting product E Biotin AP alkaline phosphate c Molecular Beacon i Nonradioactive method for detecting speci c sequences of nucleic acids ii Typlical molecular beacon probe is 25n long 1 15nt in the middle are complementary to the target DNA and are designed so that this single stranded molecles does not form a structure in which these nucleotides base pair with one another 2 Snt at each end are compementaw iii Fluorophore is attached to the 539 end and a quencher is attached to the 339 end d DNA Fingerprinting i Repeating unit is 9bp there are 5 6 and 7 repeating units per cluster quot Distinguishing individuals with DNA analysis is called DNA ngerprinting iii Two Methods 1 Minisatellide DNA a Minisatellite DNA is individuals speci c and chances to nd in two individuals are very rare b Sample DNA is digested with restriction enzymes agarose gel electrophoresis transfer onto nylon membrane hybridization c Southern blot of a forensic DNA sam e d DNA samples from the victim the defendant39s shirt and the defendant were treated with the same restriction enzyme e Here th ebanding pattern of the DNA extracted from the blood on the defendant39s shirt is identical to the victim39s DNA banding pattern and different from the defendant39s pattern f The sizes of the DNA molecules in these bands are estimated by comparison with the positions of the sizing standards DNA ampli cation with PCR e Random Ampli ed Polymorphic DNA RAPD i Arbitraw oligonucleotide primer is around 910bp ii Because of the small size this primer will bind at many places on chromosomal DNA iii Amplification of intenening by PCR will occur when two of the oligonucleotides on opposite strands are oriented facing one another and are 1003000bp apart iv Advantages 1 The same universal set of oligonucleotide primers can be used for all plant species 2 No genomic libraries radioactivity Southern transfers or DNA hybridization reactions are required so a large number of samples may be easily and rapidly characterized 3 The process can be automated f Poly Acwlamide Gel Electrophoresis PAGE i Ethidium bromidestained bands following PAGE of PCRamplified plant DNA ii Three separate oligonucleotides were used to amplify fragments from each of the two cultivars iii Cultivars 1 and 2 show identical patterns of bands with oligonucleotides A and C iv However they have different patterns when oligonucleotide B is used hence oligonucleotide B can be used to distinguish between cultivars 1 and 2 v Advantages 1 Same set of nucleotides 2 No genomic libraries Radioactivity method g RealTime PCR SYBR Green Dye i Labeling the DNA that is ampli ed in a PCR with a uorescent dye and monitoring the uoresence that results when the dye bound to a double stranded DNA is ittadiated with light of a certain wavelength it is possible to watch the production of the PCR products SYBR green doesn39t bind to the ssDNA however it binds to dsDNA as synthesized iii Dye binds to doublestranded ampli ed DNA iv Bound DNA shows uoresence h Antigen Detection by ELISA i mmunoquantitative PCR ii Upon adding the second antibody it binds to a different epitope on the antigen the bound antigen is visualized by the action of alkaline phosphate bound to the second antibody which turns a colorless substrate into a colore product iii Instead of alkaline phosphate a streptavidinbiotin complex links the second antibody to a 246bp DNA fragment with a known sequence iv Approximately 1000 fold more sensitive than ELISA 5 Genetic Diagnosis a SCA i SCA Genetic disease that is a result of a single nucleotide change ii Single nucleotide change in the Bglobin gene that causes SCA by chance abolishes a Cvnl restriction endonuclease site 1 A portion of the sequence of the wildtype HbA and sicklecell HbS human Bglobin gene The amino acids numbered from the Nterminal end of the peptide chain encoded by this ortion of the DNA are shown above the DNA se uence 2 PCR ampli cations of the portions of the Bglobin gene containing the Cvnl recognition site that is altered in the mutant gene 3 Cvnl digestion of the PCR products he normal wildtype gene has three Cvnl sites between the PCR primers and the mutant 0 4 Size distribution fragments following gel electrophoresis of Cvnldigested PCRampli ed 3globin DNA 1 b C Interferons a 1039 F39 m 5 AA homozygous condition for the normal Bglobin gene 6 AS heterozygous condition 7 SS homozygous condition for the sicklecell anemia Bglobin gene PCROLA i OLA Oligonucleotide Ligation Assay ii Proce ure Synthesize a pair of oigonuceotide probes Hybridize probes to PCRampli ed DNA Add igase to hybridized DNA Bind probes to streptavidin wash Add antidigoxigenin antibodyalkaline phosphate conjugate wash add substrate Normal DNA will have a colored substrate by the reaction of the insoluble colored product reacting with Alkaline Phosphate 7 The mutant DNA will have a colorless substrate by the absence of Digoxygenin and Alkaline Phosphate P PIF P NT Padlock Probe i When the bases at the 539 and 339 ends of the probes are completely paired to the target DNA ligation can take place 1 When there is a single base mismatch at the 339 end of the probe ligation cannot occur and the probe umes a conformation that does not allow hybridization ii The ligated probe remains bound to the target DNA which is bound to the surface of a 96well microtiter plate The nonligated probe is removed during washin The bound probe is detected by interaction with the reporter molecules If the reporter is biotine then avidin and a biotinylated enzyme such as alkaline phosphatase are added sequentially 4 A colored well indicates the probe is present and bound to the target DNA or RNA 9 quot Natural proteins produced by the cells of the immune ssystem of most vertebrates in response to challenges by foreign agents such as viruses parasites and tumor cells Interferons belong to the large class of glycoproteins known as cytokines Types i Type II 1 Binds to IFNGR 2 In humans this is IFNv ii Type I 1 All type I IFNs bind to a speci c cell surface receptor complex known as the IFNo receptor IFNAR that consists of IFNAR1 and IFNARZ 2 Type I interferons present in humans are IFNo IFNB and IFNu Type III 1 Signal through a receptor complex consisting of IL10R2 also called CRF24 and IFNLR1 also called CR 12 Protocol to Isolate IFN cDNA i Sizefractioned mRNA was isolated from human leukocytes reverse transcribed and inserted into the PstI site of plasmid pBR322 The approximately 6000 clones that were produced following transformation of E colwere divided into 12 pools of 512 clones each Pools of clones rather than individual clones were tested to speed up the identi cation process iii The plasmid DNA from each pool was hybridized to a crude IFN mRNA preparation iv The input mRNA that hybridized to the plasmid DNA was separated from the cloned DNAmRNA hybrids and translated in a cellfree protein synthesis system v Each translation mixture was then assayed for IFN antiviral activity 1 The pools that showed IFN activity contained at least one clone with a cDNA that hybridized to IFN mRNA vi Positive pools were divided into eight subgroups of 64 clones each and retested 1 This subgrouping process was repeated until a clone with a complete cDNA for a human IFN was iden le Interferon Gene Shuf ing i Comparison of the sequence of the IFNo2 and IFN03 genes shows shared restriction enzyme sites RE1 REZ and RE3 Digestion of the genes at the indicated restriction sites and ligation of the resulting fragments generate a number of different hybrid IFN genes of which four possibilties are shown Hybrid IFNs generated greater activity than the parent molecule 1 Some hybrid genes can induce cells to express 239539 oigoisoadenylate synthase 2 This enzyme generates 239539 linked 39 39 u ti at later cellular gtcleaves viral RNA f Pharmaceuticals i Human Growth Hormone Produced in E coli approved for human use 2 Also binds to the prolactin receptor Sitespeci c mutagenesis to prevent this binding Tumor Necrosis Factor 0 1 Antitumor agent 2 Toxic and nonspeci c TNFo fusion construct with a tumor cell targeting peptide Interleukins 1 Control in ammatory responses Hard to administer as therapeutics Probiotic administration of IL10 to control Crohn39s disease 2 Human Growth Hormone hGH Somatotropin 191amino acid 22125 Da b Native and Modi ed i H treatment is given to the chronic renal insuf ciency and Turner39s syndrome ii Oligonucleotidedirected mutagenesis Hist 18 21 and Glu174 was altered used to alter hGH so that it no longer bound to the prolactin receptor but retained its speci city for the growth hormone receptor iii Limitation of hGH native cross reactivity with other cell tyes iv Every day therapy is required inconvenient and expensive c Growth Hormone B39n 39ng 39 Derivatization of growth hormone by coupling it to a protein of the growth hormone receptor using a 20 amino acid pepti e Monomeric derivative 2 Dimeric derivative 3 Monomeric derivative bound to a growth hormone receptor 4 Stays in the system for longer time of perio s Therapy required every 10 days 3 Tumor Necrosis Factor TN F Alpha a umor Necrosis Factor Alpha i Antitumor agent ii Toxic and nonspeci c 1 TNFo fusion construct with a tumor cell targeting peptide iii Addition of 6amino acids didn39t alter receptor binding site however enhanced antitumor activity and demonstrated less toxicity 2 4 Enzymes a Cystic Fibrosis i Most common Caucasian genetic disease ii Results from loss of chloride channel function in plasma membrane Alters the physiology of the mucus layer in the lungs iv Generates massive lung functions Breathing difficulties due to pathogen bio lm formation v Neutrophils phagocytes recruite vi Neutrophils and pathogens both killed 1 amounts of DNA released 2 Extremely viscous mucus 3 Breathing difficulties b DNase I i Hydrolyzes long polymeric DNA into short oligomers ii Puri ed enzyme delivered by an inhaled mist into lungs of CF patients 1 Decreases viscosity of the mucus 2 Relieves most severe smptoms iii Problem lysed neurophiles release actin Actin binds an inhibits Dnase I reduces effectiveness c Alginate Ly se i Alginate 1 Polysaccharide polymer produced by seaweed and many bacteria 2 Chains of BDmannuronate and oLGuluronate 3 Crosslinking with Ca2 and metal ions gtelastic gel ii Pseudomonas aeng nosa quotmucoidquot strains 1 oduces copious amounts of alginate in biofilms 2 Increases viscosity of mucus in CF patients Falobacter um sp 1 Produces an alginate lyase 2 Cloned and expressed in E coI39 iv Alginate LyaseProducing Clone An alginate lyaseproducing clone from a clone bank of a Falobacter um so In E co39 2 The alginate that is present in the growth medium is digested by alginase secreted by E coI39 clones that the alginate in the vicinity of this colony is not crosslinked when Ca2 is adde v Recombinate Falobacter um alginate lyase protein precursor in E coI39 a peptide is removed from the Nterminus of the 69kDa precurosor to yield a 63kDa protein that can depolymerize alginate from both seaweed and bacteria 2 A second cleavage even converts the 63kDa protein to a 23kDa protein that is active againsr sea ed alginate and a 40kDa protein that hydrolyzes bacterial alginate vi DNA Construct Encoding the 40 kDa Alginate Lyase 1 Baams subtlI39s expression vector The leader peptide from a B subtlI39s alphaamylase gene is fused to the Nterminus of the lyase coding sequence 3 Under control of the penicillinase promoter 4 oduces activate lyase in me 39um d Enzymatic Conversion of Phenylalanine i 39 se phenylketonuria results from the impaired function of enzyme PHENYLALANINE HYDROXYLASE ii Treatment strategyconrolled diet of PHENYLANALINE or SEARCH for another enzyme 5 Intestinal Interleukin10 Secreting Bacteria on In ammatory Bowel Disease in Mice 6 0 ukIns i Control in ammatory responses in Crohn disease and Ulcerative colitis ii Hard to administer as therapeutics because IL10 is not clinically acceptable as this needs to be injected in frequent in39ections Prebiotic Lactococcus lad395 administration of IL10 to control Crohn Disease Data Mouse IL10 De cient mouse Ulcerative Colitis Mouse 5 o Dextran Sulfate which in ames the intestine Ulcerative colitis 39Mouse IL10 de cient mouse engineered IL10 producing L Prevented onset of ulcerative colitis lad395 Mouse 75 dextran sulfate engineered IL10 producing L 50 reduction in ulcerative colitis act395 Problems with monoclonal Abs a Generated by mouse fusion cell lines i B Cells Ab production and B cell myelomas growth ii Problem Mouse antibody crossreactivity in humans iii Answer Humanmouse fusion cell ines 1 Chromosomes of human B cells are unstable in fusion cell lines 2 Ethical to intentionally inject humans and collect splenic cells 3 Human myelomas cannot replace mouse B cell myelomas iv An alternate v Chimeric or Humanized Mab Structure of Ab H and L contains contain variable VL and VH and constant CL CH1 CH2 and CH3 domains with their CDRs CDR1 CDRZ and CDR3 b The Fv Fab and Fc portions of an antibody molecule are delineated c The Nterminal NHZ and CTerminal COOH ends of each polypeptide chain are indicated d CDR ComplementarityDetermining Region Engineered Chimeric Ab a he VL and VH DNA regions from the immunoglobulin L and H genes that encode part of a mAb were substituted or the VL and VH DNA regions of a human Ig molecule b The product of the constructed gene is a chimeric partially humanized Ig with the antigenbinding speci city of the mAb and both lowered immunogenicity in humans and human Fc effector capabilities Engineered Humanized Ab he CDTs CDR1 CDRZ and CDR3 from the genes for H and L Ig chains of the mAb replace the CDRs of the genes for a human antibody b The product of this constructed gene is an Ig with the antigenbinding speci city of the mAb and all the other erties of a human antibody molecule Human Monoclonal Antibodies and Geno Mouse ouse Ab genes are inactived by speci c deletions in Embryonic Stem ES cells which are subsequently used to generate transgenic mice unable to make b The human genes encoding Ig light and heavy chains are introduced on a YAC into mouse ES cells c These cells are used to generate transgenic mice able to synthesize both mouse and human Abs N E 5 5 9quot d The mice generated from these two types of manipulation are crossbred and mice that can synthesize only human re selected immunized and used to make hybridomas producing human A s 11 Generation of Combinatorial Library of VL and VH regions of the Ab in E coI39 a Steps i Extract mRNA from isolated lymphocytes ii Convert mRNA to cDNA iii Amplify H and L chains by PCR iv Cut with speci c set of restriction endonucleases 1 Clone H chain sequences into quotH Chainquot bacteriophage vector and or 2 Clone L chaIn sequences into quotL chainquot bacteriophage vect v Combine H and L chains into one bacteriophage vector vi Screen plaques from Ag binding vii Excise H and L chains as part of a plasmid viii Transform E coI39 with plasmidHL chain DNA construct Ix H nest Agbinding Fv fragment from E CDI DNA Constructs of an Fv Combinatorial Gene Libra a Portions of the cDNAs of the LA and HB chains are separately cloned into bacteriophage A vectors b Each of these libraries is digested with EcoRI and then fragments from the H chain library are ligated to the fragments from the L chain libraw Fv Ab Combinatorial Library in the Bacteriophage M13 e cDNAs for the VL and VH regions are ampli ed by PCR and then ligated to DNA that encodes a short linker ide b Each singlechain antibody DNA construct is cloned into gene 3 of bacteriophage M13 There are three copies of the phage gene 3 protein which is a phage surface protein per M13 bacteriophage Construction of a Large Library of SingleChain A B cells from several nonimmunized individuals were collected and pooled the mRNA was isolated and used to program the synthesis of cDNA oligonucleotide primers containing DNA sequences that included small portions of the framework region sequence were added to the cDNA preparation and all six CDRs were ampli ed separately by PC b The ampli ed CDRs from all six PCRs were mixed together with oligonucleotides encoding the framework regions and the linker and genes encoding the variable L and H domains were synthesized by overlap extension PCR MabBased Drug Delivery Systems a Cancer quotVaccinesquot Immunotoxins i Toxins 1 Catalytic A subunit often causes death 2 Cell binding B subunit responsible for host cell speci city ii Immunotoxins 1 Catalytic A subunit causes cell death 2 Recombinant quotBquot subunit antibody that recognizes cancer protein ligand for an upregulated cancer receptor Antibody as Therapeutic a Anti brin antibody monoclonal Ab that is speci c for the brin found in blood clots is coupled to plasminogen activator PA b After the complex binds to the brin of a blood clot the plasminogen activator causes plasmin to accumulate in the vicinity of the clot c The plasmin then degrades the clot Antisense RNA a A speci ed mRNA which prevents translation of the protein anti sense RNA To be an effective therapeutic agent an antisense RNA must bind to a speci ed mRNA and prevent translation of the protein c One that base pairs with a speci c mRNA is called an antisense oligonucleotide d Inhibition of translation of speci c mRNAs by antisense As nucleic acid molecules i The promoter and polyadenylation regions are marked p and pa respectively the intron is indicated by the letter A and the exons are indicated by numbers 1 and 2 1 cDNA AS gene is cloned into an expression vector in reverse orientation and the construct is transfected into a cell where the AS RNA is synthesized The AS RNA hybridizes to the target mRNA and translation is blocked An AS oligonucleotide is introduced into a cell and after it hybridizes with the target mRNA translation is locked e cDNA for human insulinlike growth factor 1 ILGF1 cloned on a vector in the antisense orientation under the transcriptional control of a metallothionein promoter MTp i Following transfection into tumorcausing cells low levels of ZnSO4 are added the cells have decreased 2 tumorigenIcI 2 Antisense Oligonucleotides ON and their Selection a The sequence speci c effectiveness of chemically synthesized antisense ON relies on hybridization to an accessible nucleotide sequence on the target mRNA resistance to degradation by cellular nucleases and ready delivery into ce 5 b ON with about 1524nt have suf cient speci city to hybridize to a unique mRNA c Potential mRNA target sites are determined by testing a set of antisense ON with cells in culture that produce the target mRNA d Those antisense ON that diminsh the translation of the speci ed protein are selected e Since ON are susceptible to degradation by intracellular nucleases i It was important to nd ways to synthesize molecules that are resistant to attack by nucleases without affecting the ability of the antisense ON to hybridize to a target sequence f Dosing Regimen of an Antisense ON i Dosing regimen of an antisense ON designed to lower LDL cholesterol ii Doses of the antisense on ranged from 50400mg per injection g Antisense oligonucleotides have been tested to determine if they can control psoriasis h A disease of uncontrolled epidermal growth that causes red scaly itchy patches to appear on the skin i IGFl has been implicated in the pathogenesis of psoriasis because IGFl receptors are presnt in excess in psoriatic lesions 3 Ribozymes Rz Rz are naturally occurring catalytic RNA molecules RNA metalloenzymes that are approximately 40 to 50 nucleotides in length and have separate catalytic and substratebinding domains b Compared with protein therapeutics an important advantage of Rz is that they are unlikely to evoke an immune response in a treated animal or human c The substratebinding sequence combines by nucleotide complementarity and possible nonhydrogenbond interactions with its target sequence d The catalytic portion cleaves the target RNA at a speci c site e For therapeutic purposes either hammerhead or hairpin ribozymesnamed for appearance of secondary structure may be i However some workers have suggested that hammerhead ribozymes are preferable because of their ability to more ef ciently recognize bind to and cleave a range of different mRNAs f Ribozymes are naturally occurring catalytic RNA molecules RNA enzymes that have separate catalytic and substratebinding domains g Deoxyribozyme 1023 RNasemRNA Substrate Complex i Arti cial no natural deoxyribozymes known ii Bene ts much more stable than RNA 100000 fold iii BUT can39t be synthesized by cell it39s DNA iv Must be delivered directly to cells 4 Aptamers a Aptamers are nucleic acid sequences RNA or DNA that bind tightly to proteins amino acids drugs or other molecules b Typically 1540 nucleotides long Aptamers are attractive as potential therapeutic agents i Because of their high speci city ii Releative ease of production iii Low or no immunogenicity iv Longterm stability d SELEX Procedure i DNA is transcribed into RNA ii Folded RNA aptamers with 539 and 339 anking regions mixed with target molecules form aptamertargeted complex iii Dissociate aptamertarget complex iv Reverse transcribe selected aptamers v PCR amplify aptamer cDNAs vi Rescreen selected aptamers against target molecules to nd highaf nity aptamers 5 Interfering RNAs RNA interference RNAi is a system within living cells that helps to control which genes are active and how active they are b Two types of small RNA moleculesmicroRNA miRNA and small interfering RNA siRNAcentral to RNA interference c RNAs are the direct products of genes and these small RNAs can bind to speci c other RNAs and either increase or rease their activity for example by preventing a messenger RNA from producing a protein 6 DNA Construct of a SingleChain Antibody Against the Hemorrhagic Septicemia Virus G Protein PCW TGFBsignal peptide VH variable domain from mouse hybri oma heavy chain i Spacer 42nt encoding a 14amino acid spacer connecting VH and VL VL variable domain from mouse hybridoma light chain CL constant part of the human light chain gene 7 Examples of Targeting Systems a gulation of muscle growth and development by myostatin in nontransgenic mice A and in transgenic mice B that overexpress the protein follistatin b Cholesterol and siRNA A conjugate of cholesterol and an siRNA in which the cholesterol is coupled through the 539 OH of the sense strand of the siRNA 1 The cholesterol facilitates uptake of the siRNA into speci c tissues a The antisense strand becomes part of the RISC and specifies where the mRNA is to be cleaved c Use of Nonpathogenic Strain of E co39 to deliver siRNAs to certain tissues i The bacterium was engineered to produce the protein invasin which permits E co39 to enter Blintegrin positive mammalian cells as well as the gene HyA encoding listeriolysin O which permits the shRNAs synthesized by the bacterium to be released inside the mammalian cell 1 Types of Vaccines a Prophylactic i Prevent or ameliorate the effects of a future infection by any natural or quotwildquot pathogen b Therapeutic i Cancer vaccine Gardasil c Vaccines containing killed inactivated microorganisms which have been killed with chemical or heat i Flu Cholera Bubonic Plague HepA d Vaccines containing live attenuated virus microorganisms disabled virulent properties e Subunit a fragment of the microorganism can create an immune response f Peptide vaccine g Innovative i Conjugate 1 Bacterial have polysaccharide outer coats linking outer coats to proteins eg toxins the immune system can be led to recognize the polysaccharide as if it were a protein antigen ii Recombinant Vector 1 Combining the physiology of one microorganism and the DNA of another iii DNA Vaccination 2 Subunit Vaccine a Animal virus i Generally consist of a relatively small nucleic acid genome 3200kb of either ds or ss DNA or RNA within a viral protein capsid Development against HSV 1 HSV is cloned to form cloned viral glycoproteinD gD gene 2 gD gene is transfected into CHO cells grown in culture they secrete gD proteins 3 gD proteins purified 4 Adv Vaccines are safe and stable with less side effects 5 Dis expensive to prepare may be less effective iii Foot and Mouth Disease 1 Viral RNA to cDNA 2 Clone cDNA to form a cDNA for VPA fused to M52 replicase gene which forms immunogenic fusion protein 3 VP1 Viral Protein 1 4 M52 bacteriophage is an icosahedral bacteriophage with a d2734nm with an pI of 39 can be propagated in E coli commonly at ATCC 15597 3 Limitations of the Peptide Vaccine a To be effective epitope must consist of a short stretch of contiguous AA b Peptide must be able to assume the same conformation as the epitope in the intact viral particle c A single epitope may not be sufficiently immunogenic 4 DNA Vaccine a Created from an infectious agent39s DNA b Nonpathogenic S flexneri to deliver foreign DNA to mammalian epithelial cells i Wild type asd bacterium is pathogenic ii Mutant asd deletion mutation encodes betasemialdehyde dehydrogenase bacterium is not pathogenic 5 6 N W P Transformed mutant with plasmid DNA bacterium is not pathogenic and delivers plasmid DNA Advantages of Genetic Immunization over Conventional Vaccines a Cultivation of dangerous infectious agents NOT required b Since genetic immunization does not utilize any viral or bacterial strains there is NO chance that an attenuated strain will revert to virulence c Since no organisms are used attenuated organisms that may cause disease in young or immunocompromised animals will not be a problem d Approach is independent of whether the microorganism is difficult to grow or attenuate e Production is inexpensive because protein does not need to be produced or purified f Storage is inexpensive because of the stability of DNA g One plasmid could encode several antigensvaccines Attenuated Vaccines a Contain live attenuated virus micobes live microbes that have their virulent properties disabled b Depleting part of the Cholera Toxin i Plasmid with A1 peptide clal and xbal ii Intact gene separated Xbal with T4 DNA ligase brings them together with a deleted A1 peptide DNA sequence iii Conjugation into V cholerae DNA with tetracycline resistance gene insert iv Homologous recombination v Chromosomal DNA now has deleted A1 peptide c K3L Restriction Endonucleases a Enzyme that cuts dsDNA following a restriction site in the DNA b Found in bacteria and archaea are thought to have evolved to provide a defense mechanism against invading viruses c Host DNA methylation by methylase evades restriction enzyme activity CloningSelecting Restriction Enzyme Gene a Pstl Cloning Modification Enzyme a Ddel b Vector pBR322hindlll c Plamids cloned with D desulfuricans Abbreviations to Memorize a 2KLG 2ketoLgluconic acid b 25DKG 25diketoDgluconic acid c ANS Antranilate synthase d DS 3deoxyDarabineheptulosonate7phosphate synthase e PRT Antranilate phosphoribosyltransferase f Kan39 anamycin resistance allows presence of vector to be selected in E coli g Tsr39 Thiostrepton resistance gene allows for selection of transformed Streptomyces strains h SacB gene encoding enzyme converting sucrose into cellular toxins I act actinorhodine J tcm tetracenomycin k fren frenolicin l gris griseusin m minPKS minimal polyketide synthase n KR Betaketoreductase 0 AR Aromatase p CYC Cyclase q 7ACA 7aminocephalosporanic acid r 7ADCA 7aminodeacetoxycephalosporanic acid s DAOC deacetoxycephalosporin C 5 Enzymes to Memorize a Tryptophanase converts tryptophan to indole b Naphthalene dioxygenase converts indole to cisindole23dihydrodiol derived from NAH plasmid c Xylene oxidase converts indole to indonyl derived from TOL plasmid d ANS Converts Chorismate to Anthranilate e PRT Converts anthranilate to indole3glycerol phosphate f DS Converts phosphoenol pyruvate and erythrose4phosphate to 3deoxyDarabine heptulosomate7phosphate g Serine acetyltransferase Converts Lserine acetylCoA to Oacetylserine M at 256 changed h sopenicillin N synthase catalyzes synthesis of isopenicillin N from DLalphaaminoadipylL cysteinylDvaline i DAmino acid oxidase converts Cephalosporin C to ketoAD7ACA gene from fungus F solani j Cephalosporin acylase converts cephalosporin C GL7ACA and ketoAD7ACA to 7ACA gene from bacterium P diminuta k Tyrosinase converts tyrosine to DOPA 34dihydroxyphenylalanine l Catechol oxidase converts DOPA to OQuinine m 3Ketothiolase converts acetylCoA n AcetoacetylCoA reducase converts acetoacetylCoA to D3hydroxybutyrylCoA o Poly3hydroxybutyric acid synthase D3hydroxybutyrylCoA to poly3hydroxybutyric acid p Sorbitol dehydrogenase converts Dsorbitol to Lsorbitol 1 Retroviral Vectors a Cleavagestage embroys usually at the eightcell stage are infected with a defective retrovirus carrying a transgene b Implanted females give birth to transgenic pups c ADV Effective d DIS lt8kb pieces of DNA may lack essential regulatory sequences e Retroviral contamination helper strain retrovirus may also incorporate into genome 2 DNA Microinjection Method a Eggs are obtained from donor females that have been induced pregnant mare39s serum and human gonadotrophin hormone to superovulate i Superovulated female produces approx 35 eggs normally 510 b Mated with males killed fertilized eggs were flushed c Microinjection was performed in fertilized eggs d Male pronucleus enlarged 3 P U1 9 N e Purified samples of transgene construct are microinjected into the male pronucleus of a fertilized egg f After the female nucleus completes meiotic division to become a female pronucleus karyogamy nuclear fusion occurs g Implanted females give birth to transgenic pups h ADV several hundred pronuclei can be injected in one day 2540 eggs are implanted in foster mother i Problems i No step 100 efficient ii 35 inoculated eggs develop into live transgenic animals 1 66 survive injection procedures 2 25 of implanted eggs develop into pups 3 25 of pups transgenic iii Not all transgenic animals are created equal 1 Wrong site of integration 2 Copy number may be too high 3 Insert into a site to disrupt a critical gene for normal phenotype Engineered ES Cell Method a ES Cell culture is initiated from the inner cell mass ofa mouse blastocyst b ES cells are transfected with a transgene c After growth the transfected cells are identified by either the positivenegative selection or PCR d Population of transfected cells can be cultured and inserted into blastocysts which are then implanted into foster mothers e PositiveNegative Selection i tk thymidine kinase HSV cells treated with gancyclovir ii Neo resistance to G418 iii Negative is nonspecific integration positive selection is HR CreloxP a ATAACTTCGTATAGCATACATTATACGAAGTI39AT b loxP comes from P1 bacteriophage c Regulation involves removal of stop codon for lacZ HighCapacity Vectors a Transgenic mice expressing human antibodies YAC transgenesis b MoAbs to treat cancer and other diseases c Rodent MoAbs are immunogenic d Hard to routinely create human MoAbs e Humanizied MoAbsonly contain 510 mouse content f Solution Create a transgenic mouse that is capable of synthesizing an extensive array of different human antibodies Senile Plaque a Extracellular deposits of amyloid in the gray matter b Deposits contain high microglia and astrocyte concentration c 4kDa protein variants ranging 3942aa d All derived from APP gene amyloid precursor protein e ApoE4 presenilin 1 and 2 gene P51 and PS2 are genes responsible for hAD and can be used to create transgenic mice Tetracycline Controlled Transcriptional Activation a Inducible expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or a derivative b Tetoff system DOX must be absent for transcription c tTA protein that binds tetO promoter and turns on transgene d Teton system DOX must be present e rtTA gene under control of cellspecific promoter 8 Mouse Model of HD a Tetoff system b Variant HD gene exon 1 with 94 CAG repeats c tTA under control of promoter active in forebrain d Dox added to water during gestation to prevent embryonic lethality e Dox removed from water after birth mutant HD expressed symptoms diappeared when Dox added back to water 9 Cloning Animals a Sheep Nuclear Transfer Method i Nucleus ofan ovum is removed with a pipette ii Cells from the mammary epithelium ofan adult are grown in culture and the Go state is induced by inhibiting cell growth iii A Go cell and an enucleated ovum are fused and then renucleated ovum is grown in culture or in ligated oviducts until an early embryonic stage iv Implanted into a foster mother where development proceeds to term b Transgenic Chickens i Cells from balstoderm donors are removed transfected with a transgene and inserted into the subgerminal space of an irradiated recipient blastoderm c Transgenic SheepGoats i Success at inserting a human TG into sheep for alphalantitrypsin d Cow i Annie producing lysostaphin kills Staph aureus 10 Benefits of Using Proteins in Milk a Milk is renewable b Can be collected wo harming animal c Novel drug is confined to a particular compartment d Normal posttranslational processing of protein e Purification from milk is relatively straightforward 11 Steps in Development of Transgenic Cattle a Collection of oocytes b In vitro maturation c In vitro fertilization d Centrifugation of eggs e DNA microinjection into male pronuclei f In vitro embryonic development to blastocyst stage g Embryo implantation h Screening for offspring
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