Experimental genetics - I

Transcription

1 Experimental genetics - I

2 Examples of diseases with genetic-links Hemophilia (complete loss or altered form of factor VIII): bleeding disorder Duchenne muscular dystrophy (altered form of dystrophin) muscle disorder Cystic fibrosis (altered form of cystic fibrosis transmembrane conductance regulator (CFTR)) pulmonary disorders So, understand normal function of a gene and correlate aberrant function with altered (mutated or lack of ) form of gene product Therapy: restore gene function

3 OVERALL APPROACHES of GENE MANIPULATION 1) Gain of function (hyperactive mutant, overexpression) 2) Loss of function (dominant negative mutant, gene deletion or mrna targeting)

4 How does a deletion mutation make a protein constitutively active? -signal A B A Main effector domain B effector Unfolded structure Intramolecular association between domains A and B folds the protein hidden effector domain (AUTOINHIBITION) Inactive protein (can t bind effector) + signal Signaling molecule A B A B -signal B (removal of autoinhibition active protein) TRUNCATED form (constitutively active)

5 How does a point mutation make a protein constitutively active? Example: p Y Protein is active only tyrosine is phosphorylated Replace Y(tyrosine) with E (glutamic acid), COO - of E mimics ve charge of phosphate (constitutively active) E

6 Loss-of-function strategies 1) Knock down the activity of the protein of interest Gene (DNA) mrna protein Knock-out gene (cultured cells, animals) Degrade mrna (cultured cells) Inactivate protein (cultured cells) 2) Make cells express an altered form (mutated) of the protein that acts as dominant negative (i.e mutated protein overrides the effect of the wild-type version)

7 Dominant Negative action Example Case: B (a kinase) binds to and phosphorylates C phosphorylated C becomes active and causes downstream effect Kinase domain P (phosphorylation) B C B C Effect B C-Binding domain C No phosphorylation of C B* C No effect B* B* B* If [B*] >> [B] (conc) B* replaces B from binding to C B endogenous protein B* mutant protein Intact C-binding domain, but lacking kinase domain

8 Basic strategies of studying gene function Classical genetics Reverse genetics Random mutation of genome Isolate mutants that give rise to Phenotypes Isolate the gene(s) responsible for phenotype (GENETIC SCREEN) Functional analyses of the gene product (protein) (Phenotype to Genotype) Isolate the gene of interest Gene manipulation in cells /organism Analyze phenotype, functional analyses of gene product (protein) (Genotype to Phenotype) GENE CLONING

9 HOW TO ISOLATE your gene of interest? Typical Source material: mrna (represents collection of transcribed genes) so, make cdna library; c-complementary) by reverse transcription 5 cap mrna translated Reverse Transcribe 3 polya tail Reverse Transcription ss cdna will include exons, sequences corresponding to 5 and 3 UTRs; BUT NO INTRONS

10 First Step: Isolation of mrna Utilizes the fact that only mrna has polya tails Oligo dt (T ) Cellulose column mrna eluted Run cellular RNA through the column No salt wash polya-mrna hybridizes with oligo DT High salt wash mrna remains bound trna, rrna eluted

11 Second Step: Reverse Transcription (ss cdna synthesis) Oligo-dT priming (mrna mix) ss cdna synthesis +RT, Nucleotides, as primer (mrna/cdna hybrid) RT: Reverse Transcriptase Problem: In Oligo-dT priming, sometimes cdna does not reach the 5 end Solution: Use a random primer (6-10 oligonucleotide in random sequence) Thus, make cdnas of different length (from different 3 starting positions)

12 Third Step: Nicking of mrna (mrna/cdna hybrid) (Ribonuclease H) Fourth Step: ds cdna synthesis + DNA polymerase, Nucleotides, DNA ligase RNA serves as primer (ds cdna mix) cdna library

13 Fifth Step: PCR cdna of the gene of interest by using suitable flanking primers cdna mix PCR cdna of the gene of interest

14 sixth step: propagation of cdna 1) A shuttle to carry cdna 2) A mechanism to make unlimited copies of cdna

15 Plasmid based propagation Daughter cells Chromosomal DNA E. Coli bacteria Plasmid DNA (circular ds, extrachromosomal, Smaller compared to chromosomal DNA; one function is to code for proteins that confer resistance to antibiotics) Ex: β-lactamase inactivates ampicillin

16 ORI Origin of replication ( bp) Replication of plasmid DNA starts here R E P L A C A Plasmid B L E Foreign DNA can be inserted here Antibiotic resistance gene (ampr/kanr) amp amplicillin kan - kanamycin Elements of a plasmid DNA

17 Multiple restriction sites TAAGAATTCAAGCTTCC Ecor1 Hind3 (example) MCS MCS multiple cloning site (also called polylinker region) (ampr/kanr) ORI Plasmid Cloning vector (Genetically engineered plasmid) (restriction enzyme sites introduced in the nonessential region)

18 Restriction digest of plasmid DNA and Ligation of cdna Inserted cdna Cloning vector ORI (ampr/ kanr) Cloned cdna

19 Transformation of E. Coli with plasmid DNA E.Coli Introduce plasmid DNA into E.Coli by heat shock and CaCl 2 (Transformation) Transformed with cdna Fail to transform

20 Selection of Transformants Criterion: Growth in antibiotic containing environment Plate E. Coli on antibiotic (Amp/kan) containing growth media - transformants will survive and mutiply untransformed E. Coli will die transformed untransformed hr Tranformant colony (individual clone) plate Agar +nutrient+ Antibiotic 37 o C [All identical transformants in a colony] Isolate plasmids from clones, DNA sequencing to Confirm right clones

21 Now you have a cloned gene Reverse genetics Isolated protein with known structure Isolate the gene of interest STEP 1: ACCOMPLISHED!!! Gene manipulation in cells / organisms Analyze phenotype, functional analyses of proteins (Genotype to Phenotype) STEP 2: GENE MANIPULATION 1) Gain-of-function 2) Loss-of-function

22 Overexpression - (introduce more copies of DNA encoding your protein of interest into cells) gene ORI Cloning vector Excise and transfer Strong Promoter Elements (TATA + Enhancer) ORI (ampr/kanr) MCS Mammalian Expression vector Poly A signal (neo R /hyg R ) Cloned cdna in a Plasmid vector Elements of an expression vector Mammalian selection markers neo R neomycin resistance gene hyg R hygromycin resistance gene

23 PCR the gene w/ indicated primers (with built in restriction sites at ends) GAATTC TTCGAA ori Cloning vector neo R 5 3 GAATTC CTTAAG AAGCTT TTCGAA 3 5 EcoR1 site Hind III site GAATTC CTTAAG a b PCR product AAGCTT TTCGAA

24 GAATTC CTTAAG a b PCR product AAGCTT TTCGAA Cut PCR product with EcoR1 and HindIII AATTC G Cut expression vector with EcoR1 and HindIII GAATTC CTTAAG MCS AAGCTT TTCGAA A TTCGA G CTTAA AATTC G AGCTT A A TTCGA ori Original Expression vector neo R

25 G CTTAA AGCTT A Ligation of cut vector and PCR product + AATTC G A TTCGA ori GAATTC CTTAAG AAGCTT TTCGAA Expression vector Carrying your gene neo R Cloned overexpresser Transform E. Coli with new plasmid, select transformants purify more plasmids from bacterial culture

26 Introduction of expression vector into cells (TRANSFECTION) Plasmid DNA+ anionic lipid liposome Step 2: cells + liposome +ve charged liposome Fusion of liposome with plasma membrane Plasmid DNA(-ve charged) Entry of plasmid DNA LIPOSOME mediated transfection Alternative techniques: electroporation (electrical pulse creates pores in cell membrane, DNA gets in); microinjection

27 Transient vs Stable TRANSFECTION Chromosomal DNA Plasmid DNA (episomal) Transient (~20-70% positive) Upto 1-2 weeks untransfected Select with neomycin (analog: G418; kills untransfected cells) *** neo R offers resistance to G418 Integration of plasmid into chromosome Stable (100% positive)

28 Mutations (point or deletion) PCR-based point mutagenesis let s say you want to change a particular histidine (CAC) to arginine (CGC) ori CAC GTG Original Expression vector PCR w/ overlapping primers incorporating the base pair change CGC GCG Strand 2 Strand 1 Original Expression vector neo R ori neo R Note: PCR products will incorporate base change

29 Products CGC CAC mutated GCG mutated GTG PCR synthesized Strand 1 Original Strand 2 (bacterial) PCR synthesized Strand 2 Original Strand 2 (bacterial) Methylated? NO YES NO YES Degrade original strands by an enzyme which cuts ONLY methylated DNA CGC GCG mutated Transform E. Coli with new plasmid, select transformants, purify mutated plasmid from culture transfect into cells

30 ori let s say you want to delete a portion of the gene of interest (from point b to c in the figure below) a b c PCR w/ indicated a Primers a b c PCR-based deletion mutagenesis Original Expression vector (with built in restriction sites at ends) neo R ori GAATTC TTCGAA b Strand 2 Original Expression vector neo R GAATTC EcoR1 site AAGCTT Hind III site GAATTC CTTAAG a b PCR product AAGCTT TTCGAA

31 GAATTC CTTAAG a b PCR product AAGCTT TTCGAA Cut PCR product with EcoR1 and HindIII AATTC G Cut expression vector with EcoR1 and HindIII GAATTC CTTAAG MCS AAGCTT TTCGAA A TTCGA G CTTAA AATTC G AGCTT A A TTCGA ori Original Expression vector neo R

32 G CTTAA AGCTT A Ligation of cut vector and PCR product + AATTC G A TTCGA ori GAATTC a CTTAAG a b b AAGCTT TTCGAA Original Expression vector neo R Cloned deletion Mutant Transform E. Coli with new plasmid, select transformants purify mutated plasmid from culture transfect into cells