Knock-out animals and Transgenic animals

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Knock-out animals and Transgenic animals

• ES cells

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Embryonic stem (ES) cells
Pluripotent stem cells derived from the inner
cell mass of the blastocyst
Can be cultured, manipulated and then reinjected
into blastocysts, where they can go on to
contribute to all parts of embryo.
In principle, ES cells also might be able to
generate large quantities of any desired cell
for transplantation into patients.
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Totipotent and pluripotent cells
Totipotent meaning that its potential is
total.
isolated directly from the inner cell mass of
embryos at the blastocyst stage.
(IVF-IT surplus embryos in case of humans)
pluripotent they can give rise to many types
of cells but not all types of cells (no fetus
developed).
www.laskerfoundation.org/ news/weis/estemcell.html

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Adult stem cells multipotent but not totipotent
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Stem cell cultures
- LIF
LIF (leukaemia inhibitory factor) maintains stem
cells in an undifferentiated state
ES cells spontaneously differentiate when
allowed to aggregate in the absence of LIF
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Human stem cell lines available
(August 28, 2001)
http//www.the-funneled-web.com/images/Embryonic2
0stem20cells.gif
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KNOCKOUT MICE
Isolate gene X and insert it into vector.
Inactivate the gene by inserting a marker
gene that make cell resistont to antibiotic
(e.g. puromycin)
Normal () gene X
Genome
Defective (-) Gene X
Transfer vector with (-) gene X into ES cells
(embryonic stem)
VECTOR
e.g.(NeoR)
MARKER GENE
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Vector and genome will recombine via homologous
sequences
From Yankulov lectures
Genomic gene
Exon 1
Exon 2
Exon 3
Exon 4
Homologous recombination and gene disrution
Grow ES cells in antibiotic containing media
Only cell with marker gene (? without target
gene) will survive
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Problems with homologous recombination
Unwanted random non-homologous recombination is
very frequent. This method provides no selection
against it
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From Yankulovs lectures
Replacement vectors
Gene segment 1
Gene segment 2
NeoR
HSVtk
Linearized replacement plasmid
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Inject ES cells with (-) gene X into early
mouse embryo
Transfer embryos to surrogate mothers
Resulting chimaras have some cells with ()
gene X and (-) gene X.
Mate them with normal mice
Lucky you, if germline contain (-) gene X
Screen pups to find -/ and mate them
Next generation will split as 31 (Mendelian)
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Problems with interpretation of knock-out
experimets
1 ) Knockout kills early embryo. How to estimate
effect of adult? 2) No phenotype. Redundancy or
just subtle change? 3) Variable phenotype 4)
Combinatorial action of genes the pinball
model. .
Knock-outs by themselves are not enough to tell
you what your gene does to every orgen
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Some answers
Many knock-out embryos die because of placental
insufficiency (failure of vascular interface)
Grow them on transplanted normal placentas !!!
Study ENU-mutated animals as additional approach
Create conditional knock-outs !!!
Will be discussed after Knock-ins (as you have to
produce knock-in first in order to make
conditional knock-out)
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Random mutagenesis to study animal genes and
functions
Dominant mutations will show up in 1st
generation of progeny. Recessive mutations
need to breed F1 progeny with wildtype mice,
then intercross the F2s or backcross F2s with
their father.
From Dr. J. Martin Collinson
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GOOD and BAD sides of in vivo mutagenesis

• Mutagenesis screens are phenotype-driven.
• 2. No a priori assumptions about
• what genes are involved in the organ system you
  want to study.
• .
• 3. Lots of mice.
• 4. May miss mutations.

Can reveal interesting mutations in known genes
that would not have been tried otherwise !!!
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To produce transgenic animal we have to introduce
full-size gene construct
ATG
promoter elements ORF
(incl. transcription start)

SV40 polyA signal
Intron could be removed
Various factors involved with the design of the
transgene or what happened when it integrated
mean that different mice containing the same
transgene may show different expression levels
or patterns.
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Knock-in animals
Microinjection in fertilized eggs
Transformation of ES cells
ES cells are selected by Neomycin (Neo accompany
Your Gene)
Transformed ES cells are injected into 3 day
embryo (blastula)
The transgene is injected into the male
pronucleus of a fertilized egg
Chimerae etc as for knocks
The DNA is inserted in the genome RANDOMLY by
non-homologous recombination
G0 offsprings from surrogate mothers contain
transgene in ALL cells
G0 crossed with non-transgenics. Offsprings
called FOUNDERS
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DNA transfer into the egg vs. ES cell
transformation

• ES cell technology works well in mice only.
• Other transgenic animals are produced by egg
  injection

2. Injection of eggs is less reliable (viability
of eggs, frequency of integration), but it helps
to avoids chimeric animals
3. ES approach provides more control of the
integration step (selection of stably
transfected ES cells)
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Transgenic mice
The growth hormone gene has been engineered to be
expressed at high levels in animals.
The result BIG ANIMALS
Mice fed heavy metals are 2-3 times larger
metallothionein promoter regulated as heavy
metals
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antifreeze gene promoter with GH transgene in
atlantic salmon
GH gene comes from larger chinook salmon
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Wild and domestic trout respond differently to
overproduction of growth hormone.
So in some cases, GH not effective.
From Yankulov
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Problem with GH fish
Transgenic salmon will escape from fisheries and
breed with strains in the wild ??? If the
transgenic fish have a mating advantage (not
clear) and are less fit (which they are), their
offsprings will produce negative effect on the
normal population.
Solutions 1) To grow sterile fish 2) To grow
fish inland without chances to escape in the wild
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Conditional knock-outs
inactivate a gene only in specific tissues and
at certain times during development and life.
Your gene of interest is flanked by 34 bp loxP
sites (floxed).
If CRE recombinase expressed
Gene between loxP sites is removed
From Dr. J. Martin Collinson
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How to FLOX a gene
1.Electroporate targeting vector into ES cells,
followed by /- selection
NeoR/ HSVtk- cells selected
TK
NeoR
loxP
loxP
loxP
2. transiently express Cre and select for ES
cells that lose neomycin resistance.
After step 2 cells could be either
NeoR- cells selected
knock-out
floxed
Make mice and breed floxed allele to
homozygousity.
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3. Mate FLOXed mice with mice carrying a Cre
transgene
Marker gene
Promoter elements Cre IRES GFP
SV40 p(A)
intron
Crucial element. Your recombinase would be
expressed in accordance with specificity of your
promoter. Promoter could be regulated
!!! artificailly or naturally
From Dr. J. Martin Collinson
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Tet-on and Tet-off systems
Reminder!!!
Now we have 3 transgenes in the same mouse
1. Tetracycline Transactivator (tTA) with
constitutive promoter
2. CRE recombinase with Tet-regulated promoter
3. Your gene with loxP sites for CRE
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Tet-on and Tet-off systems
Reminder!!!
http//gweb1.ucsf.edu/labs/conklin/Images/fig2tTA.
gif
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The Tet regulatory system
TET does not need an uptake system TET is an
established and safe drug TET regulation is
tight and sensitive There is an extensive
knowledge-basis for improvements Regulation
works in most organisms when properly
constructed Extensive experience in bacteria
lower/higher eukaryotes
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Tamoxifen inducible system
4-OH-tamoxifen a fake estrogen used as an
anti-estrogen to treat breast cancer
Special CRE used (Called Cre-ER)
a fusion of Cre with a mutated form of the
estrogen receptor that no longer binds estrogen
but DOES bind tamoxifen.
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Cre-ER is activated after addition of tamoxifen
Cre-ER-TX Dissociates from Hsp90
Cre-ER goes to nucleus And removes Floxed gene
GENE IS INACTIVATED
TAMOXIFEN added
NO TAMOXIFEN
Get control of Cre both from the promoter and
from topical addition of tamoxifen or by
injection of TXF into pregnant mothers
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Cre-mediated transgene activation
Introduction of a small piece of interrupting
nonsense into a transgene that can be removed by
Cre to allow production of transgene product
Nonsence with stops (Floxed)
Cross this transgenic mouse with one expressing
Cre in tissue of interest. In cells where Cre
is expressed and located in nucleus, get.
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More about stem cells
Embryonic stem cells
Adult stem cells
Truly pluripotential
More restricted pattern of differentiation
several countries have sanctioned deriving human
ES-cell lines from surplus embryos created
through in vitro fertilization
medical gain without ethical pain
although several human ES-cell lines have been
made, they will not be immunologically
compatible with most patients who require cell
transplants.
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More problems with ES cells (not only ethics)
1) although several human ES-cell lines have been
made, they will not be immunologically
compatible with most patients who require cell
transplants.
2) undifferentiated ES cells form teratomas after
implantation in the body (should be completely
differentiated in vitro)
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Compare ES cells and MAPC (multipotent adult
progenitor cells)
Stuart H. Orkin and Sean J. Morrison
Jiang et al.
The expression of Oct-4 in ES cells correlates
with their versatility (should be high in MAPC,
if they are true versatile)
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Human in vitro fertilization
http//www.stanford.edu/dept/ GYNOB/rei/pics/scan9
.tif
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Polar Body Sampling
Polar bodies
Polar body
Meiosis II Requires fertilization
Meiosis I
primary oocyte
secondary oocyte
zygote
To test for disease gene carried by mother, DNA
from first polar body (or both the first and
second polar bodies) can be tested. If the first
polar body contains only the disease allele, the
oocyte would contain only the normal allele, and
the oocyte would be used for IVF. Conversely, if
the polar body contains the normal allele, the
oocyte would contain the disease allele and
would be discarded.
Removal of polar body
From Yankulov
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Blastomere Isolation
After IVF, 1-2 blastomeres can be removed from
the 8-cell embryo without doing any harm.
These cells can be tested by PCR, and only
clean embryos lacking disease alleles will be
transferred into the uterus.
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http//www.faseb.org/opar/cloning/cloning.htm
From student presentation Aman Arya, Nancy Chen,
Dan Perz, Dave Reichert, Ronnie Wong
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Nuclear Transplantation
Nucleus comes from someone to be cloned
1. Enucleation of the cell
2. Nuclear transfer
removal of the nucleus
chromosomes are gently sucked out with a sharp
micropipette
A. electrofusion
whole donor cell injected beneath the zona
pellucida (the outer membrane of the oocyte)
and fusion of cells induced by electrical
impulses
From the a mature unfertilized oocyte (egg) Or
from the cell in quiescent state (inactive G0
phase of cell cycle) OR metaphase II
B. nuclear injection
naked nucleus microinjected into cytoplast
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Electrofusion
http//www.brinkmann.com/pdf/cell_fusion.pdf
fusion pulse
Cells brought close together
Heterokaryon phase nuclei distinct
fusion product
Fusion induced by electric pulse
From student presentation Aman Arya, Nancy Chen,
Dan Perz, Dave Reichert, Ronnie Wong
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Genetic Reprogramming
If cell for cloning taken from adult organism
de-differentiation rearranging the genome of
the nucleus to restore its totipotency so it can
differentiate into different types of cells and
develop into a whole organism must occur after
nuclear transfer to successfully produce the
clone required for the nuclei from adult cells
to develop normally best completed in
unfertilized oocytes (as plasma donors)
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Re-programming never achieved with same success
as fertilization
Fig. 5 from Nature Reviews Genetics 3 671
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Development of the embryos from cell with alien
nucleus
may be induced by chemical treatments developing
embryos are grown in a culture to assess their
viability
Implantation of Embryo
embryos are surgically transferred into the uteri
of suitable surrogate mothers many embryos are
transferred to each surrogate mothers to ensure
implantation
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Mammal Cloning Timeline
Megan and Morag
Dolly
http//www.cnn.com/2001/WORLD/europe/08/06/clone.c
ritics/index.html
From student presentation Aman Arya, Nancy Chen,
Dan Perz, Dave Reichert, Ronnie Wong
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Tetra
http//hs.houstonisd.org/hspva/academic/Science/Th
inkquest/gail/text/benefits.html
From student presentation Aman Arya, Nancy Chen,
Dan Perz, Dave Reichert, Ronnie Wong
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Dolly with her surrogate mother
Dolly

• Born in July 1996 at the Roslin Institute in
  Scotland
• First mammal to be cloned from an adult mammal
  using the nuclear transfer technique
• 277 attempts were made before the experiment was
  successful
• Dolly died in February 14, 2003 of progressive
  lung disease at the age of 6 whereas normal
  sheep can live up to 12 years of age.

Dolly with her first newborn, Bonnie
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Mammal Cloning allows propagation of endangered
species
http//www.howstuffworks.com/cloning.htm/printable
January 8, 2001 Noah, a baby bull gaur, became
the first clone of an endangered animal.
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Comparison of Cloning Success Rates in Various
Animals
The table shows success rates of cloning when
mature mammal cells were used.
Yanagimachi, R.  2002.  "Cloning experience from
the mouse and other animals." Molecular and
Cellular Endocrinology.  21 March, 187.
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Development and survival of cloned mouse embryos
Majority of the embryos die before and after
implantation. This figure shows that the present
cloning technique is highly inefficient.
Yanagimachi, R.  2002.  "Cloning experience from
the mouse and other animals." Molecular and
Cellular Endocrinology.  21 March, 187.
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Clone Birth Defects

• Cloned offspring often suffer from large
  offspring syndrome, where the clone and the
  placenta that nourished it are unusually large.
• Cloned offspring often have serious inexplicable
  respiratory or circulatory problems, which causes
  them to die soon after birth.
• Clones tend to have weakened immune systems and
  sometimes suffer from total immune system
  failure.
• Very few clones actually survive to adulthood.

Clones appear to age faster than normal. Clones
experience problems associated with old age, such
as arthritis, while they are still young. This
may be due to the fact that clones have shorter
telomeres
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The whole story about cloning is not a
reproductive story
The possibility of using cloning technology to
grow organs genetically identical to our own for
transplantation thereby avoiding rejection of
foreign issues
Greatest danger Clones being harvested for
their body parts
http//medlib.med.utah.edu/WebPath/CVHTML/CV001.ht
ml
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http//easyweb.easynet.co.uk/sfl/rlb3a.jpg