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Lecture 19: Recombinant DNA Technology

and Reverse Genetics

Restriction analyses, nucleic acid probes used to detect mutant genes
in hereditary disease!
Recombinant DNA technology produces large amounts of medically
important proteins

Used to treat

Epidermal growth factor
Factor VIII
Fibroblast growth factor
Follicle stimulating hormone
Growth factor -1
Lung surfactant protein
Serum albumin
Tissue plasminogen activator

cystic fibrosis
Growth 'disorders'
Cancer , Aids
Cancer , Rheumatoid arthritis
Respiratory distress
Plasma supplement
Heart attack

Molecular causality of an inherited medical problem, first described by A. Garrod in

1900, illustrating a modern forward genetic analysis that starts with mutant
variants, in this case related to alkaptonuria see pp 230 -234.

In 1898, this rather brilliant

doctor, Archibald Garrod,
showed homogentisic acid is
excreted into the urine of
alkaptonuria patients (black
urine disease). He then
provided the first evidence of
an inherited (inborn) error
(mutation) in metabolism !

In 1902, Garrod found that black urine disease

was prevalent in a few families and showed it
was inherited as a Mendelian recessive,
autosomal allele. In 1908 he hypothesized it
was due to an an inherited enzyme deficiency.
Investigation into the nature of this deficiency
languished until fifty years later, when it was
proposed that HGO, the enzyme that splits
homogentisic acid was absent in the liver of
patients with alkaptonuria.

In 1992, the gene for HGO was mapped by
recombination studies to 3q2.


In 1995 a gene with similar activity was

isolated in Aspergillus nidulans, a mould
(fungus). They used this sequence to do a
computer search of fragments in a human
cDNA library and found a gene with 52%
sequence similarity (homology).

Functional ID test 1: The human cDNA
>DNA showed HGO activity in bacteria and

Sequence ID test 2: (liver activity): when used
as a probe, it detected a single RNA generated
in liver cells .

Mendelian Test 3 (chromosome location):

The cDNA clone was used as a probe on
chromosomes, it bound to 3q13 (120,628,167
- 120,682,570 bp).

The cDNA clone was then used to recover the
full length gene in a genomic library. It had
14 exons and was more than 60,000 bases long.

Mendelian Test 4: is the proposed role of
HGO consistent with the inheritance of
black urine disease ?

A family of 7 where 3 of the children suffered
from alkaptoneuria was tested for mutations.
PCR was used to amplify each exon, and they
were sequenced. One parent had a
proline>serine substitution at position 230 in
exon 10, the other a valine to glycine
substitution at position 300 in exon 12.

All the affected children were PV300; G230S: suggesting mutations at these 2 sites inactivate

Fernndez-Can JM, Granadino B, Beltrn-Valero de
Bernab D, et al. The molecular basis of alkaptonuria. Nat
Genet 1996;14:1924.

You have the DNA, now put it into

Eukaryotic organisms.

Transgene insertion (organism) The introduction of

foreign DNA into a eukaryotic organism.
Transformation is
often used to
describe uptake of
DNA by any
organism. Soaking
cells in cold salt
solutions is effective
with some bacteria.
Similar treatments
may work on yeast,
but not most plant
or animal cells.

and (5) you can use transposable

Viruses as cloning vectors in mammals

Animal viruses such as retroviruses may prove useful vectors for
recombinant gene therapy to treat single gene disorders. !

Using retroviruses as animal vectors and

GENE THERAPY-A brief history!
1990: 4 year old Ashinti de Silva was
successfully treated for severe combined
Immunodeficiency disease (SCID) by
somatic cell transformation of mutation
in the blood enzyme adenosine
deaminase. She is still being treated.!
2000: Later trials on a group of 10
patients treated for SCID, produced
mixed results. Unfortunately two of the
patients had a (retroviral) insertion into
a site that caused acute leukemia, leading
NIH to the terminate gene therapy
experiments on humans. In 2003 China
opened a human gene therapy clinic.!
2009 - On November 11, 2009 Amsterdam Molecular Therapeutics announced that it
has successfully treated Duchenne muscular dystrophy (DMD) in an animal model
with its proprietary gene therapy.

Human Genome Project Information

What factors have kept gene therapy from becoming an effective treatment for genetic disease?
* Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any
condition, the therapeutic DNA introduced into target cells must remain functional and the cells
containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic
DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from
achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
* Immune response - Anytime a foreign object is introduced into human tissues, the immune
system is designed to attack the invader. The risk of stimulating the immune system in a way that
reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's
enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in
* Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy studies,
present a variety of potential problems to the patient --toxicity, immune and inflammatory responses,
and gene control and targeting issues. In addition, there is always the fear that the viral vector, once
inside the patient, may recover its ability to cause disease.
* Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the
best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as
heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the
combined effects of variations in many genes. Multigene or multifactorial disorders such as these
would be especially difficult to treat effectively using gene therapy. For more information on different
types of genetic disease.

In the 1990s it was discovered that the most effective way of

introducing foreign genes in mammal model systems was
microinjection .

When you put

foreign DNA
into a host
determine the
pattern of its


Injecting naked
double stranded DNA
or using a vector to
transport it.
Ectopic insertion random insertion
into a chromosome
during the pronucleus
..(or mostly not),


Chromosome insertion through

microinjection into a pronucleus
stage of an egg/embryo

Mus musculus transgenes:

Ectopic insertion of linearized After fusion of the pronucli, the
transgenic DNA may (randomly)
integrate (ectopic insertion)

Possibly causing :
(1)Ideally, the transgene integrates
into a germ line chromosome ,
and is expressed.!


(2) But insertion could have a

position effect on expression
due to the absence of normal
regulatory sequences nearby
(3) Insertion may cause internal
rearrangements of the native
chromosome, or the foreign
array, possibly causing mutation
and unexpected phenotypes!

Genetic transformation of mice, suggesting gene therapy.

1% recovery
5,000 copies


Domestic animals with

enhanced growth rates
sounds like an economic
Transgenic pigs:
increased growth rates
but only on high protein
Females were sterile and
both sexes were lethargic.


Example 2: Manipulate the DNA, insert it into a plasmid with a

selectable marker, that will alter the transformed nematodes
phenotype in some detectable way, inject vector into C. elegans.

Two possibilities,
(1) ectopic integration
into a chromosome
(position effect ,
insertion effect) ? or
(2) it may be
maintained as an


Example 3 - Genes into Drosophila

9 nuclear division
(multinucleate) embryo

Rosy is

(1)Bacterial (vector) plasmid

carrying a P element with DNA
of interest inserted into the
transposase sequence (i.e.
disabled) with the 31 bp terminal
inverted repeat sequences within
the vector plasmid.
(2) The helper plasmid with a
complete transpose enzyme, but,
no terminal inverted repeat



What makes this very useful is that

you know the (inverted repeat
flanking sequences.
Extract the DNA, fragment it,
separate the fragments on a
Southern then
Use PCR primers that contain the
(inverted) terminal repeat
sequence, amplify outwards the
intervening segment and
sequence it. Use this sequence
to identify the neighboring
sequence where it has inserted.

Inserting transgenes into a nonmodel organism


Example 5: recombinant plants

The bacteria Agrobacterium tumefaciens naturally transforms some
plant species when the T-DNA segment of the 200kb Ti plasmid
inserts into a chromosome. This ability is used to produce
recombinant plant cells.

The foreign gene is spliced or inserts into T-DNA. This intermediate is

recombined into a disarmed plasmid (no ori with a selectable marker)
introduced by Agrobacterium infection.! The insert is indicated by a linked


selectable marker -antibiotic (e.g.

kanamycin) resistance gene.!
Screening cells :!
1) Functional -nopaline production
2) Insert Kanamycin resistance!
3) Test Sequence- Southern probe
hybridization for T-DNA or the
4) The flanking TDNA sequences where
the transposon integrated can be
sequenced outwards- possibly
indicating where on the genome it
OK - Induce the callus cells to grow into
a plantlet.!

Insert genes with a virus, ectopically, use

transposable elements - quasi random
insertion ."
Or, target a gene, disrupt its function and
screen for an altered phenotype indicating its function- Reverse Genetics

Producing a targeted gene knockout: modifying a gene (with an

unknown) function!
Use transposons to insert & disrupt gene
function (in Drosophila).!


(1) Use T DNA to insert in Plant genomes!

(2)Target a mouse gene and look for phenotypic
consequences using a breeding design!

(3)Antisense RNA stops expression, by annealing

to the native mRNA, then through the RNAi
Reverse Genetics
pathway. Make the antisense strand by cloning
(temporary knockout) the gene, excise the ORF, and reverse the coding
sequence.Use RNAi with a portion of the gene
of interest in inverse orientation, a hairpin
bound by RISC (RNA induced Silencing
Complex) and degraded. !


The SALK Homozygote T-DNA Collection
This project specifically addresses an important aim of the 2010 project - to identify genetically
stable loss-of-function mutations in all Arabidopsis genes. Computational analysis of the
initial genome sequence of Arabidopsis thaliana in 2000 provided evidence for the existence of
approximately 25,500 genes. .The identification of T-DNA/transposon insertion mutations
in all Arabidopsis genes has been an on-going aim of worldwide functional genomics program for
Alonso et al. 2003, Science 301Over

225,000 independent Agrobacterium

transferred DNA (T-DNA) insertion events in the genome of the reference plant
Arabidopsis thaliana have been created that represent near saturation of the gene
space.The precise locations were determined for more than 88,000 T-DNA
insertions, which resulted in the identification of mutations in more than 21,700
of the 29,454 predicted Arabidopsis genes. .Insertion mutations were identified
in genes that are regulated in response to the plant hormone ethylene.!

Targeted Mutations in mice: Inducing sequence- specific

mutations without knowing the phenotype
Vector with 2 markers:
Neomycin resistance NeoR and
Ganci(y)clovir sensitivity (tk+)


Engineer a functional Neomycin gene (mutant

sequence) targetting your sequence of interest



Vectors can be designed to cause specific mutations

in genes which can be selected in cell cultures. In this case:
(a) targeting
(b) vector ectopic
(c) no insertion

This design depends on the difference between

homologous recombination and random insertion