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.
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
Adult stem cells multipotent but not totipotent
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
Human stem cell lines available (August 28, 2001) http://www.the-funneled-web.com/images/Embryonic%20stem%20cells.gif
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
Vector and genome will recombine via homologous sequences From Yankulov lectures Genomic gene Exon 4 Exon 2 Exon 3 Exon 1 Homologous recombination and gene disrution Grow ES cells in antibiotic containing media; Only cell with marker gene ( without target gene) will survive
Solution: Replacement vectors The knock-out construct contains the 1) NeoR gene flanked by 2) two segments of the target gene and 3) the HSVtk gene Part of the gene replaced with NeoR ES cells are selected for integration of NeoR and against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir Problems with homologous recombination Unwanted random non-homologous recombination is very frequent. This method provides no selection against it
From Yankulov’s lectures Homologous recombination Random integration NeoR NeoR+/ HSVtk- NeoR+/ HSVtk+ HSVtk will convert gancyclovir into a toxic drug and kill HSVtk+ cells Replacement vectors Gene segment 1 Gene segment 2 NeoR Linearized replacement plasmid HSVtk
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 3:1 (Mendelian)
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
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)
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
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 !!!
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.
DNA 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
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)
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
antifreeze gene promoter with GH transgene in atlantic salmon GH gene comes from larger chinook salmon
Wild and domestic trout respond differently to overproduction of growth hormone. So in some cases, GH not effective. From Yankulov
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
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 loxPsites (floxed). If CRE recombinase expressed Gene between loxP sites is removed From Dr. J. Martin Collinson
Gene of interest 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.
3. Mate FLOXed mice with mice carrying a Cre transgene Marker gene Promoter elementsCre 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
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
Tet-on and Tet-off systems Reminder!!! http://gweb1.ucsf.edu/labs/conklin/Images/fig2tTA.gif
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
Tamoxifen mimics the action of estrogene and binds to estrogen receptor (ER). Complex of Cre-ER With Hsp90 In cytoplasm Floxed gene in the nucleus GENE IS ACTIVE 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.
Complex of Cre-ER With Hsp90 In cytoplasm Floxed gene in the nucleus GENE IS ACTIVE 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
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….
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.
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)
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)
Human in vitro fertilization http://www.stanford.edu/dept/ GYNOB/rei/pics/scan9.tif
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
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.
Biotechnology of Mammalian Cloning Embryo Splitting • earliest method of cloning • success limited to embryos split before implantation • Parthenogenesis • only possible in females to give female progeny • still investigating – so far mostly failed attempts • Nuclear transplantation • main technique in current cloning experiments http://www.faseb.org/opar/cloning/cloning.htm From: student presentation Aman Arya, Nancy Chen, Dan Perz, Dave Reichert, Ronnie Wong
ENUCLEATION cytoplast oocyte 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
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
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)
Re-programming never achieved with same success as fertilization Fig. 5 from Nature Reviews Genetics 3: 671
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
1984 – A live lamb was cloned from sheep embryo cells Mammal Cloning Timeline 1986 – Early embryo cells were used to clone a cow 1993 – Calves were produced by transfer of nuclei from cultured embryonic cells 1995 – Two sheep, named Megan & Morag, were cloned using embryo cells 1996 – Birth of Dolly, the first organism to be cloned from a fully differentiated adult cell 1997 – Transgenic sheep named Polly was cloned containing a human gene Megan and Morag Dolly http://www.cnn.com/2001/WORLD/europe/08/06/clone.critics/index.html From: student presentation Aman Arya, Nancy Chen, Dan Perz, Dave Reichert, Ronnie Wong
1998 – 50 mice were cloned in three generations from a single mouse Tetra 1998 – 8 calves were cloned from a single adult cow, but only 4 survived to their first birthday 1999 – A female rhesus monkey named Tetra was cloned by splitting early embryo cells. 2000 – Pigs and goats reported cloned from adult cells 2002 – Rabbits and a kitten reported cloned from adult cells http://hs.houstonisd.org/hspva/academic/Science/Thinkquest/gail/text/benefits.html From: student presentation Aman Arya, Nancy Chen, Dan Perz, Dave Reichert, Ronnie Wong
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
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.
Species Number of oocytes used Number of live offspring Notes Mouse 2468 31 (1.3%) - Bovine 440 6 (1.4%) 2 died Sheep 417 14 (3.4%) 11 died within 6 months Pig 977 5 (0.5%) - Goat 285 3 (1.1%) - 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.
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.
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