Screens & Recombinant DNA

Techniques in Neuroscience
October 20, 2010
Kelsey Clark

Today

Review basics of genetics

Manipulating DNA
Genetic screens
Mapping mutations
Genomic libraries
Human genetics
Measuring gene expression

Genetics 101 Review

Chromosomes

Chromosomes are made of DNA

Genes are the functional unit of DNA

Genes code for proteins

The central dogma: DNA to RNA to protein

Turning DNA to mRNA

Only Exons

Genetic code: from mRNA to amino

acid

Regulation of gene expression

RNA
Transcription
Factor
RNA
Polymerase

Promoter

nucleus

cytoplasm

GENE

How many genes?

Originally thought to be
100,000
Human Genome Project:
20,000-30,000
Coding regions: ~1.7-5%
We share 97% of genome
with chimps

Human

~25,000

Drosophila

~14,000

C. elegans

~19,000

Mouse

~29,000

Rat

~30,000

Pufferfish

~31,000

Rice

>45,000

Mutations, polymorphisms and

alleles
Mutation: change in DNA sequence due to error or
damage during replication
Polymorphism: naturally occurring variation in DNA
sequence seen in >1% of population (eg: mutation
thats been around for a while)
Allele: one version of a polymorphism

Types of mutations
Point Mutations
Silent: switch nucleotide but not
amino acid
Missense: amino acid switch
Nonsense: stop codon =
truncated protein

Frameshift Mutations
Insertion
Ex: the cat can sit !
the fca tca nsi t

Deletion
Ex: the cat can sit !
the atc ans it

Types of Polymorphisms
SNPs - single nucleotide
polymorphism (point mutation)
VNTRs - variable number tandem
repeats (microsatellites)
Transposon - mobile element that
can move around within the genome

What its all about

Identifying DNA sequences to
- identify the genes behind mutant phenotypes
- probe gene expression
Manipulating DNA to examine
- the role of a gene in cells or behavior (via knock-outs,
RNAi, inducible expression, etc)
- the role of a group of cells in behavior (by selectively
killing, silencing, or controlling activity in these cells)

The tools

Cloning (mass production)

Vectors (storage)
Restriction digest (cut)
Ligation (paste)
Gel electrophoresis (isolation by size)
PCR (amplification, modification)
DNA synthesis
Sequencing
Hybridization (find your DNA)
Transfect into cells

What is this cloning?

Identify gene of interest
Map gene
Determine DNA sequence
Make copies in bacterial clones

But wait
Cloning (mass production)

Q: How can I get bacteria to

make copies of my DNA
for me?
A: Put it in a vector

Vectors: the Storage System

Key features
Ability to replicate
Selectable marker(s)

Bonus features
Multiple cloning site
IRES (Internal
Ribosome Entry Site)
Insert DNA here

Different types of vectors have

different features
A cloning vector:
pBluescript

Features:

origin of replication
Amp resistance
Multiple cloning site (MCS)
LacZ gene

A mammalian expression
vector: pcDNA3

Features:
Amp resistance
MCS CMV promoter
polyA tail
neo resistance

IRES allows single mRNA to

code for multiple proteins
IRES = Internal Ribosome Entry Site
TK

Gene

IRES

Gene 2

NeoR

TK

FOXP2

IRES

eGFP

NeoR

Artificial Chromosomes for

Large Pieces of DNA

BACs, YACs, PACs, HACs

Features:
Centromere & telomere regions

But wait
Cloning (mass production)
Vectors (storage)
Q: How do I get the DNA from my gene into a
plasmid vector?
A: Youll need three tools:
cutting DNA with restriction enzymes
pasting pieces together with a DNA ligase
separating pieces of DNA with gel electrophoresis

Restriction Enzymes cut DNA

Recognize specific short DNA
sequences
Cleave the DNA at these sites
in a specific way

Restriction enzymes leave

various types of ends
Sticky

Blunt

Ligation:
paste ends of DNA together

Gel Electrophoresis:
separate DNA fragments by size

DNA moves through gel based on size

Can be identified by location (size)

Putting your gene into a vector

excise your gene from source and cut
vector with compatible restriction enzyme
gel purify your fragments
mix fragments & ligase
transform E. coli with ligation products
grow plate of transformed E. coli colonies
purify plasmid DNA from many colonies
for DNA from each colony, perform
restriction digest and run gel to see if it has
your gene inserted and in the right direction

Two+ days in the life of a cloner

Digest (hours)
Gel purify (hours)
Ligation (hours or overnight)
Transform (hour)
Plate cells (grow overnight)
Pick colonies (grow hours)
Miniprep colony DNA (hours)
Digest (hours)
Run gel (hours)

But wait

Cloning (mass production)

Vectors (storage)
Restriction digest (cut)
Ligation (paste)
Gel electrophoresis (isolation by size)

Q: How do I get the DNA for my gene in the first place?

A: PCR.
Q: What if my DNA doesnt have restriction sites at
each end?
A: PCR.

Polymerase Chain Reaction

(PCR): Amplify DNA
dNTPs
DNA polymerase
5

Template DNA

primers
Multiple Cycles

Exponential amplification requires only

small amounts of template

Things you can do with PCR

Make copies of a DNA
fragment
Make cDNA from mRNA
(RT-PCR)
Measure quantity of DNA
or RNA (qRT-PCR)
Modify DNA sequences
Add restriction sites
Introduce mutations

RT-PCR:
Reverse
Transcription
PCR
dNTPs
Reverse Transcriptase
3

5
3

primers

Template mRNA
DNA polymerase

dNTPs
3

5
3

Template cDNA

primers
Multiple Cycles

Adding Restriction Sites

5-GAATTC-3

dNTPs

DNA polymerase
5

Template DNA

primers
Multiple Cycles

GAATTC
CTTAAG

3-GAATTC-5

CTTAAG
GAATTC

Put recognition sequence on the ends

of the primers

Site-directed Mutagenesis
Put the mutation in
the primer

Primers

Short single-stranded oligos

Bind to ends of sequence to amplify

But wait

Cloning (mass production)

Vectors (storage)
Restriction digest (cut)
Ligation (paste)
Gel electrophoresis (isolation by size)
PCR (amplification, modification)

Q: Where do I get those primers you said I needed for

PCR?
A: They can be made via chemical DNA synthesis.

DNA synthesis

But wait

Cloning (mass production)

Vectors (storage)
Restriction digest (cut)
Ligation (paste)
Gel electrophoresis (isolation by size)
PCR (amplification, modification)
DNA synthesis
Q: How do I know what primer
sequence to order?
A: You choose based on prior DNA sequencing.

DNA sequencing

Example: Sequencing
Genomes
Using BAC
libraries & brute
force sequencing
methods we got
the genome
sequenced
shotgun
sequencing

Q: How can I be sure my DNA/RNA is present in a

sample?
A: Hybridization!

Northern/Southern blots
Southern (DNA)

Northern (RNA)

Western (protein)

In situ Hybridization
To localize mRNA in tissue (in situ)
1. Hybrid strands of mRNA
(probes) are
synthesized

ANRmENEG
GENEmRNA

GENEmRNA

2. Hybrid RNA strands are

labeled with a
chromogenic enzyme or
radioactive element
3. Brain slices are exposed to
the probes, which mark
the location of neurons
that produce the target
protein

But wait

Cloning (mass production)

Vectors (storage)
Restriction digest (cut)
Ligation (paste)
Gel electrophoresis (isolation by size)
PCR (amplification, modification)
DNA synthesis
DNA sequencing

Q: I want neurons in a living, behaving mouse to

express my gene. Right now its just sitting in E. coli.
How do I get my DNA construct into my cells of
interest?
A: Transfection.

Transfection: getting your DNA construct into

cells (or animals)

Physical
Microinjection
Gene gun
Electroporation

Chemical
Calcium phosphate
Lipid-based transfection

Viral infection

Genetic Screens

Common animal models

Species
C. Elegans

Advantages
Simple multi-cell organism, known
neural connectivity

Disadvantages
Invertebrate
Very primitive nervous system

Can readily manipulate genes

Easy forward screening

Drosophila

Short lifespan

Invertebrate

Can readily manipulate genes (in

whole organism and tissues of
interest)

Primitive nervous system

Easy forward screening

Mice

Complex higher order organism

(mammal)

Long life span and

reproduction time

Can manipulate genes

Genetic manipulations are

time-consuming and costly

Nervous system homologous to

humans

Human

Species we care most about

Complex behavioral phenotypes

Long life span and

reproduction time
Cannot personally manipulate
genes

Ways to create mutants

1) Chemical mutagenesis
2) Irradiation mutagenesis
3) Insertional mutagenesis
- insert small DNA
sequence into genome
via transposon,
disrupting normal gene
sequence

Forward and reverse genetics

Forward screen
Process of
interest

mutate

Find gene

ask the genome what matters

Hypothesis generating

Reverse screen
Gene of
interest

mutate

Effect?

what does this one do?

Hypothesis-testing

Forward screen

Choose an assay
Characterize wildtype
Mutagenize germ line
Screen offspring for abnormal
phenotype
Complementation tests and mapping
Clone the gene to mess with it in more
sophisticated ways

Example forward screen: Clock

wildtype

G1 #25

Vitaterna et al Science 1994

Complementation test:
is my mutation in the same gene as mutation X?
Homozygote of
your (recessive)
mutation

Homozygote of
recessive gene X
mutation

Offspring:
one copy of your mutation,
one copy mutation X
Wildtype?
Your mutation not in gene X

mutant phenotype?
Your mutation is in gene X

From mutant to gene:

mapping your mutation

T to C in putative
transcription factor

Recombination frequency

Linkage analysis
1) Cross your mutant with other individuals of different
phenotypic markers whose genetic locus is known
2) Score co-occurrence in offspring to see whether traits
are inherited together (linked) or independent
3) Do this with many different known genes, and you get an
approximate location for your new mutation

58

Restriction Fragment Length Polymorphism

(RFLP) analysis
RFLPs are polymorphisms in
genome that alter/destroy/create
restriction sites
Calculate frequency of
recombination between your
mutation and restriction-site
altering SNPs
Can look at linkage between your
mutation and any SNP by
sequencing, but this takes more
time and money than a digest

To the genome sequence!

You now know ~distance from several

RFLP sites (density of the RFLP sites
will depend on your animal, and even
the specific area of chromosome in that
animal). Consult the genome sequence
for genes in that general area.

A typical Drosophila screen

and mapping

Screen 1000s of mutant lines for a phenotype.

Screen 100s of lines with a more precise but more time
intensive assay.
Choose one mutant to identify for further study.
Linkage analysis to find approximate location of mutation
RFLP (or SNP sequencing) analysis to narrow region down
Look at genes in region using genomic database. If mutants are
already available, order those to check for your phenotype and
try complementation.
Start sequencing the genes in your mutant strain, looking for
coding variations from the published sequence.
Clone gene, verify with rescue construct, RNAi, etc

I wish Id done insertion mutagenesis

To map an insertion mutation:
1) sequence genomic DNA using portion of
insert DNA as primer
2) run sequence through DNA database to
determine where you inserted, which gene
was disrupted
Disadvantage: insertion not as random as radiation or
chemical mutagenesis.

Genomic database resources

Ensembl: browse the genome of any sequenced organism!

BLAST: put in a sequence, get homologous sequences from all

organisms ranked by similarity

Whats a DNA library?

Lots of pieces of DNA, stored in
plasmids which allow you to isolate and
amplify each piece separately.
Can be made from genomic DNA or
from mRNA expressed in a specific
group of cells.

Making a DNA library: putting lots of

random chunks of genomic DNA into a vector
Genomic DNA
restriction digest!

Ligate!

Making a cDNA library

mRNA
cDNA

reverse transcriptase

Clock example, continued

based on genotyping of over 2400 offspring,

linkage analysis narrowed mutation area
down to an ~4 megabase region
no previously identified genes mapped to the
nonrecombinant interval containing the Clock
locus
via Southern blot, looked for overlap in BAC
library clone containing the region of interest,
and the cDNA library from an area of the brain
important for circadian behavior (SCN)
plus lots of sequencing

King et al Cell 1997

Fresh example: capsaicin receptor

Caterina et al Nature 1997

Genes in Humans
Linkage analysis
RFLP/VNTR linkage analysis
Fluorescent in situ hybridization

Royal pedigree

Twin studies

Likelihood of linkage: LOD scores

Compares the probability of obtaining pedigree results if two

markers are linked with a certain amount of recombination
between them.
Calculate the LOD for hypothetical recomb frequency .01 to .
5 : the value with the highest LOD is the most likely
LOD score of +3 = 1000:1 for linkage
LOD score of -2 = 100:1 against linkage

Fluorescent in situ hybridization

Human genetics example:

FoxP2, the language gene

Fischer et al Nature Genetics 1998

Blue: BAC clone proximal to

translocation, not seen on der2
Green: BAC clone spanning breakpoint,
see fluorescence on der7 and der2
Lai et al AJHG 2000

Lai et al Nature 2001

Gene expression
1) Quantitative real-time PCR
2) Blots
3) Microarrays
Screen entire genome for up/down regulation

Know what youre

looking for

qRT-PCR:
Quantitative REAL-TIME PCR

How microarrays work

Gene expression
1) Quantitative real-time PCR
2) Blots
3) Microarrays

In all cases, may want to examine only

a specific population of cells.
Gross dissection
Laser capture microdissection
Fluorescent activated cell sorting

Laser Capture Microdissection:

isolate specific nuclei

Fluorescent activated cell sorting

sort cells by
surface antibodies
transgenic fluorescent tag

Questions about gene expression

How does gene expression in the hippocampus differ
from that in the amygdala?
How is gene expression in Area X altered by stress/
social interaction/learning?
How does gene expression underlie behavioral variation
in genetically identical animals?
How does a mutation in one gene alter gene expression
across the genome?
How do gene expression patterns change with age?

Example: gene expression and behavior in honeybees

Whitfield et al Science 2003