BEH.109: Laboratory Fundamentals in Biological Engineering.

MODULE 3

Eukaryotic Cells as Phenotypic Indicators:

The use of RNAi to modulate gene expression

DAY 3

Monday March 31 DAY 1

Module 3 Overview & mini-lecture on RNAi Safety Orientation Sterile Technique Transfection of EGFP & p53 siRNA into EGFP expressing HeLa cells

Tues April 1 DAY 1

Module 3 Overview & mini-lecture on RNAi Safety Orientation Sterile Technique Transfection of EGFP & p53 siRNA into EGFP expressing HeLa cells

Wed April 2 DAY 2

Comprehensive lecture on RNAi with some examples Harvest transfected cells Microscope analysis & FACS analysis Analyze data

Thurs April 3 DAY 2

Comprehensive lecture on RNAi with some examples Harvest transfected cells Microscope analysis & FACS analysis Analyze data

Monday April 7 DAY 3

Introduction to the ATM, ATR, EXO1 and AAG genes Ambion and Blast session to design new siRNAs for four genes. siRNA is ordered for next experiment

Tues April 8 DAY 3

Introduction to the ATM, ATR, EXO1 and AAG genes Ambion and Blast session to design siRNAs for four genes. siRNA is ordered for next experiment

Wed April 9 DAY 4

Introduction to DNA microarrays and overview of what will happen on days 5 & 6 Transfect four new si.RNAs; cellular RNA will be isolated over the w/e Informal Presentation of FACS data by students

Thurs April 10 DAY 4

Introduction to DNA microarrays and overview of what will happen on days 5 & 6 Transfect four new si.RNAs; cellular RNA will be isolated over the w/e Informal Presentation of FACS data by students

Monday April 14 DAY 5

Label isolated RNA and hybridize to microarray slides

Tues April 15 DAY 5

Label isolated RNA and hybridize to microarray slides

Wed April 16 DAY 6

Scan microarray slides and analyze results

Thurs April 17 DAY 6

Scan microarray slides and analyze results

Patriots Day

MIT Holiday

Wed April 23 DAY 7

MODULE 3 Student Presentations

Thurs April 24 DAY 7

MODULE 3 Student Presentations

Snapshot of the next four

weeks

We will eliminate the expression of six different genes using

RNAi technology, human cells, fluorescent

proteins and DNA

microarrays

Sunlight

Pollution

Oxidation

FoodCigarette Smoke

DNA is constantly being damaged by endogenous

and exogenous agents

DNA Repair Strategies• Direct Reversal

Photolyase, Methyltransferase, Oxidative demethylase

• Excision RepairBase excision, nucleotide excision, transcription

coupled excision repair, mismatch repair

• Lesion AvoidanceTranslesion synthesis, DNA recombination

• Double strand break repairHomologous recombination, Non-homologous end

joining

DNA Repair Strategies• Direct Reversal

Photolyase, Methyltransferase, Oxidative demethylase

• Excision RepairBase excision, nucleotide excision, transcription

coupled excision repair, mismatch repair

• Lesion AvoidanceTranslesion synthesis, DNA recombination

• Double strand break repairHomologous recombination, Non-homologous end

joining

Excision Repair

Recognition

Excision

Resynthesis

Ligation

DNA Repair Strategies• Direct Reversal

Photolyase, Methyltransferase, Oxidative demethylase

• Excision RepairBase excision, nucleotide excision, transcription

coupled excision repair, mismatch repair

• Lesion AvoidanceTranslesion synthesis, DNA recombination

• Double strand break repairHomologous recombination, Non-homologous end

joining

REPLICATION FIDELITY

• How many times does the replicative polymerase have to choose the correct nucleotide during one cell division???

• Is one mistake in a million choices acceptable?

• How is fidelity achieved?

Humans who have

inefficient Mismatch Repair are

highly prone to colorectal

and other cancers!!

GC

GT

G

Mismatchrecognition

MSH2 MSH6

MLH1

PMS2

Endonuclease?

GT

EXO1

PCNA, Pol , RPALigase?

GT

DNA Repair Strategies• Direct Reversal

Photolyase, Methyltransferase, Oxidative demethylase

• Excision RepairBase excision, nucleotide excision, transcription

coupled excision repair, mismatch repair

• Lesion AvoidanceTranslesion synthesis, DNA recombination

• Double strand break repairHomologous recombination, Non-homologous end

joining

Base Excision Repair

AAG/MPG

3MeA DNA glycosylase

initiates repair of replication

blocking lesions

Human AAG enzyme

bound to substrate

(aka MPG)

The absence of the AAG enzyme renders mouse

cells very sensitive to the toxic effects of

alkylating agents that damage DNA

You will try to knock down

human AAG with siRNA!!

DNA Repair Strategies• Direct Reversal

Photolyase, Methyltransferase, Oxidative demethylase

• Excision RepairBase excision, nucleotide excision, transcription

coupled excision repair, mismatch repair

• Lesion AvoidanceTranslesion synthesis, DNA recombination

• Double strand break repairHomologous recombination, Non-homologous end

joining

The eukaryotic cell cycle

Three pronged

attack

P53 has 18 sites for modification by

phosphorylation, acetylation, sumolation

How do deficiencies in

p53

ATM

ATR

Affect human health?

p53

Li-Fraumeni Syndrome

Germ line inheritance of mutated p53 genes

Cancer Prone

THE FIRST SIGNS of ataxia telangiectasia (A-T) usually

appear in the second year of life as a lack of balance and slurred

speech. It is a progressive, degenerative disease

characterized by cerebellar degeneration, immunodeficiency,

radiosensitivity (sensitivity to radiant energy,

such as x-ray) and a predisposition to cancer.

Ataxia Telangiectasia – Cancer Prone