BEH.109: Laboratory Fundamentals in Biological Engineering. MODULE 3 Eukaryotic Cells as Phenotypic Indicators: The use of RNAi to modulate gene expression Instructor: Leona D. Samson Teaching Assistants: Jenn Cheng and Lisa Joslin With additional

1
BEH.109 Laboratory Fundamentals in Biological
Engineering. MODULE 3Eukaryotic Cells as
Phenotypic IndicatorsThe use of RNAi to
modulate gene expressionInstructor Leona D.
SamsonTeaching Assistants Jenn Cheng and Lisa
JoslinWith additional invaluable help from Lisa
Smeester and Rebecca Fry
2
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
3
The use of RNAi to modulate gene
expression Why do we want to be able to
modulate gene expression?
4
Central Dogma of Molecular Biology
5
Transcription
6
What is mRNA?
GCU Ala
7
(No Transcript)
8
Genes were first described by their mutant
phenotype e.g., Mendel described inherited
properties like wrinkled versus smooth peaslater
Bateson coined the word gene to account for
these phenotypic traits. Genes were said be
inherited in a Mendelian fashion.
9
1940s Beadle and Tatums classic experiment with
moulds established the one gene one enzyme
hyothesis
10
In the 1940s Beadle and Tatum mutated genes to
analyze biochemical pathways Mutagens? X-rays,
Nitrogen Mustards
11
Neurospora mould
12
(No Transcript)
13
1940s Beadle and Tatums classic experiment with
moulds established the one gene one enzyme
hyothesis
14
Eliminating the expression of a gene is one of
the most powerful tools in biology We can now
engineer the deletion of specific genes to probe
their biological funtion
15
Forward Genetics Phenotype Genotype Reverse
Genetics Genotype Phenotype
16
The most common method for Reverse Genetics has
been Targeted Gene Deletion
17
Number of Genes in Different Organisms
Human 30,000 genes Mouse 30,000 genes
Yeast 6200 genes E. coli 4200 genes
Phage T4 200 genes
Influenza 12 genes
18
1997 saw the sequencing of the first eukaryotic
genome. S. cerevisiae The field is changed for
ever...
19
What does knowing the sequence of all 6,200 genes
enable?
Making 6,200
Gene Deletion Strains!

20
(No Transcript)
21
Number of Genes in Different Organisms
Human 30,000 genes Mouse 30,000 genes
Yeast 6200 genes E. coli 4200 genes
Phage T4 200 genes
Influenza 12 genes
22
Mammals are diploid! Have to knock out both genes
to test the null phenotype
23
We will eliminate the expression of six
different genes using RNAi technology, human
cells, fluorescent proteins and DNA microarrays
What cells? HeLa cells What genes? Today EGFP
..enhanced Green Fluorescent Protein gene and
p53 gene as a control Next week Four
additional genes
24
The Origin of HeLa Cells
25
HeLa cells in culture
26
HeLa cells from the Nikon microscope web site
HeLa cells have been cultured continuously for
scientific use since they were first taken
from the tumor of a woman suffering
from cervical cancer in the 1950s. They have
been utilized for many purposes,
including the development of a polio vaccine, the
pursuit of a cure for diseases such as leukemia
and cancer, and the study of the cellular effects
of drugs and radiation.
27
HeLa Human cells
JONATHON PINES
REGULATION
OF MITOSIS IN

MAMMALIAN CELLS
Mitotic HeLa cell stained with
anti-Cks1 (red), anti-tubulin (yellow) and DAPI
(blue)
28
HeLa cells as you will see them
Expression of (A) b-galactosidase and B green
fluorescent protein in HeLa cells. Cells were
transfected in 6-well plates. Expression was
visualized by X-gal staining or fluorescence
microscopy 2 days post-transfection.
29
(No Transcript)
30
You will knock down the levels of the mRNA
transcripts encoding EGFPusing RNAi technology
31
So what is RNAi? RNA interference And what are
siRNAs??? Short interfering RNAs
32
siRNAs will attack gene expression at the mRNA
transcipt level
33
(No Transcript)
34
1
846
EGFP ORF
240-259
GCAGCACGACUUCUUCAAGU dTdT
dTdT CGUCGUGCUGAAGAAGUUCA
1
1182
P53 ORF
774-792
GACUCCAGUGGUAAUCUAC dTdT
dTdT CUGAGGUCACCAUUAGAUG
35
HeLa cells as you will see them
Expression of (A) b-galactosidase and B green
fluorescent protein in HeLa cells. Cells were
transfected in 6-well plates. Expression was
visualized by X-gal staining or fluorescence
microscopy 2 days post-transfection.
36
Snapshot of the next four weeks