Recombinant DNA Technology II

1
Recombinant DNA Technology II (Lodish Chapter 5.3
and 5.5) Overview DNA fingerprinting Genome-wid
e expression profiling Whole genome
hybridization Gene therapy Transgenic
mice Knockout mice
2
DNA fingerprinting
Is based on the fact that we have variable
nucleotide tandem repeats (VNTRs) such as (CA)n
Importantly, the n differs from individual to
individual
unique
unique
CACACACACACA
PCR on different samples will yield fragments of
different length
(match)

sample from crime scene
3
O.J. Simpson was tested for 5 different VNTRs
that are highly polymorphic (n varies a lot)
His DNA matched PERFECTLY 5 out of 5
times Probability 1 700,000,000 (yes, seven
hundred million) Statistically, 6 people in the
entire world have the same pattern
4
DNA microarrays analyzing genome-wide
expression patterns

• DNA microarrays consist of thousands of
  individual gene sequences bound to closely spaced
  regions on the surface of a glass microscope
  slide
• DNA microarrays allow for the simultaneous
  analysis of the expression of thousands of genes
• The combination of DNA microarray technology with
  genome sequencing projects enables scientists to
  analyze the complete transcriptional program of
  an organism during specific physiological
  response or developmental processes

5
Principle of microarray analysis
6
A real microarray
7
Cluster analysis identifies pathways that are
turned on or off
For example, B clusters all genes involved in
cell cycle regulation some go down (green
e.g., CDK inhibitors), and some go up (red e.g.,
cyclins).
8
Samples of your choice
Gene expression profiling of patients
with peridontal diseases, cancer patients
(prognosis), bacteria causing gum infections.
9
Whole genome arrays allow for the detection of
deleted or amplified sequences (comparative
genomehybridization)
Each spot represents a sequence segment of your
entire genome (not only a gene). You hybridize
fluorescently labeled DNA from a normal cell
(green) and a cancer cell (red) to the array. If
you have A 1/1 ratio, your signal is yellow.
Deletions in cancer cells are indicated by green
spots, amplifications by red spots. Blue spots
are due to non-specific background hybridization.
10
Introducing recombinant DNA into human cells
11
Stable gene integration/Gene therapy
linearize
(antibiotic)
Best example today transfect ADA gene into B-
and T- cells in modified adeno- viruses that can
infect cells but cant multiply
ADA adenosine deaminase required to make
inosine. B- and T-cells accumulate high levels of
dATP.
12
Purines are degraded to urate
Salvage pathway
GMP IMP

ADA

Catalyzed by Hypoxanthine-guanine phosphoribosyl
transferase (HGPRT)
ADA adenosine deaminase, lack of ADA causes SCID
(severe combined immunodeficiency or bubble boy
disease)
13
Transgenic mice (carry trans-geneextra gene, can
either be mutant or wild-type)
14
(No Transcript)
15
Relevant study
p53 Transgenic mice are highly susceptible to
4-nitroquinoline-1-oxide-induced oral cancer. Mol
Cancer Res. 2006 Jun4(6)401-10. Erratum in Mol
Cancer Res. 2006 Aug4(8)599. Li, Jie added.
PMID 16778087 PubMed - indexed for MEDLINE
In this study, we did a bioassay employing mice
with a dominant-negative p53 mutation
(p53(Val135/WT)) to assess whether a germ-line
p53 mutation predisposed mice toward the
development of squamous cell carcinomas (SCC) in
the oral cavity. Treatment of the mouse oral
cavity with 4-nitroquinoline-1-oxide produced a
66, 91, and 20 tumor incidence in the oral
cavity, esophagus, and forestomach/stomach,
respectively, in p53(Val135/WT) mice. In
contrast, only a 25, 58, and 4 tumor incidence
was observed in oral cavity, esophagus, and
forestomach/stomach, respectively, in wild-type
littermates (p53(WT/WT)). The most striking
difference between p53(Val135/WT) and p53(WT/WT)
mice following the carcinogen treatment was the
higher prevalence and more rapid development of
SSC in p53(Val135/WT) mice than in wild-type
mice. To identify the precise genes or pathways
involved in these differences during tumor
development, we examined gene expression profiles
of 4-nitroquinoline-1-oxide-treated normal
tongues as well as tongue SCC in p53(Val135/WT)
and p53(WT/WT) mice. Microarray analysis revealed
that dominant-negative p53 ((135)Valp53) affects
several cellular processes involved in SCC
development. Affected processes included
apoptosis and cell cycle arrest pathways, which
were modulated in both tumor and normal
epithelium. These results showed that reduction
of p53-dependent apoptosis and increases in cell
proliferation might contribute to the observed
increase in oral cavity and gastroesophageal
malignancies in p53(Val135/WT) mice as well as to
the more rapid growth and progression of tumors.
16
Discovery of the obesity gene in knock-out mice
ob-/ob-
Wild-type
The obesity gene encodes leptin, a protein
specifically expressed in fat cells that signals
to the brain that youre full after eating a meal
17
Replace exon with antibiotic resistance marker
(neomycin)
18
Several more crossings until you get homozygous
KO animals
19
Tissue specific gene knockout mice
(e.g. leptin receptor in the brain)
20
Cre-recombinase catalyzes site-specific
recombination
Modern application to generate
tissue-specific KO mice
Origin bacteriophage P1
21
Knock-down gene expression by RNA interference
transfect into cells
siRNA is targeted to the specific mRNA. The
mRNA base pairs with the complementary siRNA
strand and is subsequently degraded
mRNA
22
What you need to know Principle of DNA
fingerprinting (analysis of VNTRs). Microarray
studies allow for genome-wide gene expression
studies This technique has revolutionized
medicine and will allow for the development of
individualized diagnostics and therapies. Comparat
ive genome hybridization can identify
deletions/amplifications in cancer and other
genetic diseases. Principles of gene therapy to
restore gene function by introducing a
cloned wild-type copy into cells (best success so
far in curing bubble boy disease) Studying gene
function in animal models Transgenic mice (carry
extra gene-mutant or wt) Knockout mice have
specific gene deletions Tissue-specific gene KO
can be achieved by Cre/loxP recombination Studying
gene function in somatic tissue culture
cells Introduction of transgenes, gene KO or
gene knock-down by RNA interference (mechanism to
destroy a specific mRNA)