Genetic Manipulation Lectures 1-7

Lecture 1 – Embryonic Stem Cells

v  Embryonic stem cells are pluripotent meaning they can differentiate to become various tissues in the body

v  Pluripotent teratomas are derived from germ cells

Ø  Ovarian teratomas are usually benign and may have different types of fully differentiated tissues like hair, teeth etc  n

Ø  Testicular teratocarcinomas are malignant

§  These cells can be kept in culture as embryonal carcinoma cells and even in this state it manages to maintain pluripotency

§  Even though these cells have pluripotency these cells also have many mutations which is probably why they are tumours in the first place

·        Embryonic stem cells were derived inspired by embryonal carcinoma cells and their pluripotency

¨      The difference is that the ES cells are not mutant therefore not cancerous

v  First embryonic stem cells

Ø  Taken from mouse embryo

§  Once blastocyst stage is reached post fertilisation the inner cell mass is cultured

§  The cultured cells were found to just keep dividing into undifferentiated cells but keeping the ability to contribute to any cell of the developing mouse

§  2 parts of the blastocyst: trophectoderm & inner cell mass

·        Trophectoderm expresses Cdx2 genes

·        Inner cell mass expresses Oct4 genes – stains green

Ø  ES cells will form teratomas in adult mice but will contribute to parts of the growing embryo if reintroduced to a blastocyst

§  ES cells can be injected into the ICM of the host blastocyst and if survives that be implanted back into the mother thus giving birth to chimeric mice

·        A chimeric mouse is a mouse that is derived from 2 fertilisation events – 1st event being the original fertilisation creating the host blastocyst and the other being the fertilisation from which the ES cells were created from

·        The chimeric mouse will have ES cells which contribute randomly to all tissues

Ø  ES cells can be maintained in vitro for a long time in the right conditions

§  Leukaemia Inhibitory Factor (LIF) prevents their differentiation

·        LIF is a cytokine

·        Without LIF – the cells will differentiate into all sorts of different cell types known as embryoid bodies

v  There are certain genes that are a marker of and required for pluripotency

Ø  Oct4

§  This a transcription factor expressed in ICM cells

§  Without this gene embryos will develop only until blastocyst stage before the die because the ICM isn’t pluripotent

Ø  Nanog

§  Also found in the ICM for maintenance of pluripotency

§  Without nanog the ICM lose pluripotency and develop as extra-embryonic tissues

v  Genetic manipulation of ES cells

Ø  This is done to figure out how different genes do and to study their functions etc

Ø  ES cells have a hydrophobic plasma membrane and DNA is hydrophilic so the DNA needs to get in somehow

Ø  You can get the DNA into the cells by electroporation

§  Electroporation is done by bathing cells in a solution of DNA then decant into a metal cuvette and give them an electric shock so the wall is damaged ever so slightly so the DNA get in

§  Post electroporation you need to select which cells have taken in the DNA

§  After selection these cells can be injected into the ICM of a blastocyst and implant into a pseudo pregnant female mouse

·        To implant the female must have sex with a vasectomized male so that the uterus is vascularised to receive the host blastocyst

·        The implantation is done under GA

·        The female will then give birth to mutant chimeric mice

Lecture 2 – Mutating Genes

v  Ethics regarding genetic modification is a big issue hence why there are regulation in place for this

Ø  Project license

§  License given for those conducting any projects involving animals

Ø  Personal license

§  Everyone working on animals NEED to have this

Ø  Purpose of these licenses is because of the 3Rs

§  Reduction

·        Reduce the number of animals to the minimum for scientifically sound results

§  Refinement

·        To make sure animals experience minimal distress

§  Replacement

·        Can we replace the animal? Ie using the animals for experimental purposes necessary

·        Even before getting the appropriate licenses the experiment has to be told and determined whether it can be done in vitro, on organoids – basically any other way other than using animals

Ø  Welfare of animals is paramount for genetically modified animals

Ø  The cost-benefit is also very important when using animals

v  Purpose of genetic manipulation

Ø  To understand the genetic basis of human health and disease

Ø  To identify and analyse the roles of particular genes - over-expression and knockout

Ø  To understand the control of gene expression as genes aren’t expressed in all tissues all the time

§  When control of gene expression is lost, it can lead to cancer

Ø  To genetically tag animals or create ‘designer animals’ for further research, medical or other use. E.g. disease models

v  The importance of a gene can be realised by mutating it and seeing what it does eg CFTR knockout

v  Spontaneous mutant mice

Ø  The small eye mouse caused by Pax6 mutation

Ø  Looptail mice caused by Vangl2 mutation

Ø  Clubfoot mice caused by Limk1 mutation

Ø  The only disadvantage with using spontaneous mutant mice is that it may not be convenient to what you want to investigate

v  How to speed up mutations?

Ø  Exposure to mutagens like xrays or certain chemicals ethylnitrosourea (ENU)
or ethyl methanesulphonate (EMS)

§  ENU creates point mutations mostly by ethylating DNA base pairs during DNA replication in replicating sperm cells

§  EMS mostly turns G/C base pairs into A/T during DNA replication

§  If you give a mouse a low dose of EMS or ENU, it creates randomly distributed point mutations at low frequency throughout the genome otherwise it is toxic and mice will just die

v  Random mutagenesis

Ø  Expose male mice to a mutagen as mentioned above so sperm is affected and will have mutations

Ø  Then mate mutated mice with wild-type female mice

Ø  Then screen babies for mutation looking at phenotype

§  This way we can investigate why a certain mouse looks like that or something else etc

§  Only heterozygous problems can be identified phenotypically so the ‘normal looking’ mice must be screened for recessive mutations

Ø  Advantages of mutagenesis screens

§  Can generate mutations in tissues without a priori assumptions or knowledge about which genes are important eg if looking at diabetic mouse and you want to figure out which genes are important in maintaining pancreatic beta cells and by chance you get a mouse that doesn’t have pancreatic beta cells – you can work out the mutated gene

§  Can generate new alleles of genes that you would never have been able to make deliberately, or would never have thought of

Ø  Disadvantages of mutagenesis screens

§  Uses very large numbers of animals
➢ Wasteful - you only find what you’re looking for
➢ e.g. limb defects, and may miss interesting mutations just by not
looking at the right bit of the animal
INCREASINGLY DIFFICULT TO JUSTIFY IN MICE
Need Technology to Create Mutations in the Genes you Want

v  Gene knockout by homologous recombination in ESCs

Ø  Knockout the gene in ES cells

Ø  When you are happy the gene is knocked out, use those ES cells to make a new mouse by injecting them into blastocysts

Ø  Homologous recombination occurs during meiosis

§  When identical DNA sequences on maternal & paternal chromosomes find each other, line up and may ‘cross over’

Ø  Homologous recombination can occur accidentally in ANY cell at ANY time à at low frequency – this fact can be exploited to introduce new DNA into cells

Ø  This is the ideal but is still quite rare

Ø  Plan is to replace red DNA with the targeting vector

Ø  The flank sequences of the vector and the red DNA are identical

Ø  The targeting vector has green fluorescent protein (GFP) and neomycin resistance

§  So cells that uptake the vector DNA will become fluorescent green and they will all be resistant to neomycin

Ø  Over the next couple of days, most ES cells will expel or degrade any DNA they took up

Ø  A small proportion will integrate targeting vector into their genome - this is likely to happen randomly

Ø  A small proportion of those cells that integrate targeting vector will do so by homologous recombination

Ø  To check the integrity of vector that has been put in, ES cells must be selected with ganciclovir and neomycin

§  Ganciclovir will kill the cells with TK

§  Neomycin will kill the cells without the neomycin resistance gene

Ø  The surviving cells are selected and injected into the inner cell mass of the host blastocysts then implant into the uterus of the pseudopregnant female thus producing chimeric offspring

v  Interpretation of knockouts

Ø  Redundancy can lead to mild or no phenotype (atthe level of analysis) eg MyoD gene involved in muscle development is knocked out but mice still have good muscles

§  Therefore, gene is redundant as other genes are doing the same job

Ø  Early embryonic lethality may prevent analysis of later events
➢ Fgf4-/- Oct4-/-

·        Knocking out Oct4 causes the embryo to die at a very early stage in the blastocyst stage

Ø  Genetic background
➢ The strain of mouse you use may affect the phenotype you see

Ø  Genes don’t act in isolation so it doesn’t mean that knocking out a particular gene and its effects are universally applicable

v  Knock-ins

Ø  The use of targeting vector and homologous recombination to introduce a new functional gene at a known location in genome

Ø  Can be used to make reporter mice expressing GFP etc., or get mice expressing one gene under the control of a promoter of a different gene

Lecture 3 – Transgenic Animals

v  Transgenic animals are animals with new DNA put into them unnaturally for research purposes so that they:

Ø  Express mutant forms of the gene you want to study

Ø  Express the gene you want to study in excess amounts, or in the wrong places to study disease processes etc

Ø  Express commercially important products e.g. insulin in milk

Ø  Express genetic markers such as Green Fluorescent Protein under the control of interesting promoters

v  Requirements for building a eukaryotic transgene

Ø  Needs a promotor that will drive expression in the tissues required

Ø  Open reading frame encoding the gene needing expressed

Ø  Sequences that ensure correct mRNA processing

v  Methods for introducing transgene into animals

Ø  Direct injection via micropipette

§  Inject into male pronucleus after fertilisation of oocyte but before nuclear fusion

§  Integration post injection is random making it complicated and unpredictable

·        The random process can result in the transgene to integrate into another gene causing disruption

·        Long-term transgene expression depends on the transgene integrating with the host DNA

§  When DNA is injected into the nuclei it is free-floating DNA – the nucleus has DNA repair enzymes and they’ll see the free-floating DNA as damage to the genome and they can do 1 of 2 things

·        DNA repair enzymes can either degrade it and then its gone or it’ll make a break in the host genome and will ligate the free-floating injected DNA into the host genome

·        When DNA repair enzymes find the free-floating DNA and cause them to link together in a chain with many dozen copies before integration which could result in problems with transgene expression

Ø  Chemical transfection

§  There are transfection reagents, and its molecules will bind to DNA but they also have a hydrophobic side so they can get through the plasma membrane of the cell

Ø  Electroporation

§  Expose cells to DNA and give them an electric shock to force DNA into the cells

Ø  Infection

§  Expose cells to viruses carrying transgene DNA that will infect cells

§  The virus genome needs to be taken out so that the transgene DNA can be inserted

§  If there is functional viral particle that doesn’t have its own genome but has a gene that you want to express then viruses can be used to get that gene into cells

§  Problem with viral infection of cells is that the virus can get shut down and will affect expression so its often used to introduce a gene into bits of the mid-gestation embryo thus creating a mosaic

§  3 classes of viruses to get DNA into cells

·        Adenoviruses

·        Adeno-associated virus

·        Lentiviruses

¨      Retroviruses

Ø  They have an RNA genome and once it gets into the cell it is reverse transcribed into DNA and will express genes on it and may or may not integrate into the host genome

Ø  Retrovirus RNA that goes in will contain genes for all the proteins needed to make more infective viruses

Ø  Retroviruses have 3 main genes

§  gag à encodes proteins of nucleoprotein core of virion

§  pol à encodes reverse transcriptase, integrase etc functions

§  env à encodes surface protein components of virion

§  These 3 genes can be replaced with a transgene but gag, pol and env are needed to infect other cells so the transgene replaced RNA needs to be exposed to a cell line independently expressing gag, pol and env but they will not have the packaging signal or the flanking sequences

Ø  It will also have flanking sequences and a packaging signal

¨      Most stable integration with these viruses

v  Introduce transgene at zygote (1 cell) stage to make transgenic animal because if introduced later, you end up making a chimeric mosaic mouse

v  If transgenic animal is made à expression from transgene may not happen for several reasons

Ø  Weak promotor/lack of regulation

§  Needs adequate promotor to drive expression of the gene

§  Need another transgene to fix this issue

Ø  Copy number

§  If you don’t know how many copies there are, it makes it harder to know what to predict in expression

Ø  Position effects (site of integration)

§  If integrated to close to a strong promotor – it could have effects on the transgene

Ø  Epigenetic modification

Ø  Genetic background

Ø  Very big transgene

Lecture 4 – Conditional Knockouts

v  Conditional gene knockouts are when certain genes are knocked out using recombinase enzyme called Cre in eukaryotic cells

Ø  This method ensures that where the gene being investigated is inactivated but just in particular cells at particular times during the life of the animal

Ø  Cre is a gene and protein that comes from the P1 phage

Ø  Cre recognises a 34 base pair DNA sequence called loxP

Ø  Any piece of DNA that is surrounded by 2 loxP sites, Cre will knock out the DNA in between the 2 loxP sites and there will only be 1 loxP site left

Ø  There is a similar system that occurs in yeast

§  Instead of Cre it is Flp and instead of loxP sites they have FRT sites

Ø  Principles of conditional knockouts

§  Need to create and maintain 2 strains (lines) of mice

·        1 line of mice in which loxP sites are surrounding the gene of interest

¨      The gene is said to be floxed by loxP

·        1 line of mice expressing Cre recombinase from a tissue-specific promoter

¨      Meaning cre is not expressed in every tissue because it’s got a promoter

·        Breed the 2 together and after a couple of generations à mice with 2 copies of the floxed gene and a copy of the cre recombinase therefore the gene will only be knocked out in the tissues where cre is expressed

§  Creating the floxed mice

·        Done by conditionally targeting ES cells with a targeting vector using homologous recombination (lecture 2 notes)

·        This time the gene of interest and the neomycin resistance gene are surrounded by loxP sites

·        Then electroporate targeting vector so that recombination occurs resulting in ES cell containing the gene of interest and the neomycin resistance gene floxed by loxP then perform selection as before resulting in surviving cells (see lecture 2)

·        This can then be injected into blastocysts creating chimeras and breed them to heterozygote floxed mice

·        Now we need to get rid of the neomycin resistance gene as it may interfere with the floxed allele

¨      Neomycin resistance gene is surrounded by FRT sites

¨      Breed mice with a transgenic mouse line that expresses Flp recombinase removing the neomycin resistance gene resulting in just the floxed allele

Ø  Refinements on the cre-lox rechnique to produce mice where it develops normally until it is an adult to then inactivate certain genes to model degenerative diseases like Alzheimer’s, Parkinson’s etc

§  Put Cre on promoters that are responsive to drugs like:

·        Tetracycline

¨      Cre with promoter called tetO that is responsive to tetracycline

¨      tetA protein needs to bind to tetO to activate Cre transcription and therefore knockout the floxed gene but this does not happen in the presence of tetracycline

¨      Called tet-off system

·        Tamoxifen

¨      Normally oestrogen binds to its receptor in the cytoplasm then the complex goes to the nucleus where it can then change gene expression – this is a ligand dependent system

¨      There is a variation of cre recombinase that covalently links to oestrogen receptors but it’s a mutated version causing it to bind tamoxifen instead of oestrogen

¨      In the absence of tamoxifen, hsp90 grabs Cre-ER and keeps it in the cytoplasm

¨      Tamoxifen displaces hsp90 and now the whole complex can access the nucleus where cre-mediated recombination can take place

§  This way it turns cre on or off by putting drugs into drinking water or by injection

v  Example

Ø  You make a knockout mouse to investigate the role of gene X in the hippocampus however the mouse dies in early embryogenesis before it even has a hippocampus

Ø  This suggests that the gene of interest is important for something in early embryogenesis that the mice aren’t getting past

Ø  But you can’t investigate gene X’s role in the hippocampus

Ø  Chimeric mice are a way to get round this problem

Ø  Targeting cre expression to tissue of interest

§  Drive Cre from the promoter of a gene that:

·        Is only expressed in the tissue you want (quite rare) OR

·        Is expressed in areas that overlap with your tissue of
interest OR

·        Knock the Cre gene into a hippocampal gene

¨      Make transgenic mouse carrying Cre on the hippocampal promoter, and check expression

Ø  Transgenic Cre DNA construct contains:

§  Promoter

·        Isolating the promoter sequence that drive Cre in tissues where you want the gene to be knocked out is a difficult task

·        This problem is solved when knocking cre in by homolgous recombination

§  Cre

§  Internal Ribosome Entry Site (IRES)

·        Viral sequence allowing ribosomes to bind to mRNA and initiate translation without scanning the 5’ nontranslated region

§  GFP or LacZ – needed to check expression

§  Intron

§  Polyadenylation signals

v  Chimeras

Ø  How to make them

§  Take the 8-cell stage embryo of mice that have heterozygous mutants for the gene being investigated and aggregate them with wild-type mice embryo and then implant them into a surrogate mother, so mosaic offspring is born

§  Alternatively, you can inject mutant ES cells into a wild-type blastocyst

Lecture 5 – Genome Editing

v  Zinc-finger nucleases (ZFN)

Ø  Normal function

§  Protein motif that bids 3 base pairs of DNA

§  Loops of amino acids held together by zinc making the zinc finger

§  Different Zn-fingers bind different 3 base pairs to control the regulation of different genes

§  They are specific to a transcription factor

§  Works in vitro or vivo with good specificity and works on any animal

Ø  Experimental ZFN

§  Linked to DNA nuclease called Fok1

§  Fok1 break DNA and so when broken DNA is repaired there is likely to be mutations present

·        Faulty repair = non-homologous end joining

·        2 Fok1s are needed to break the double stranded DNA, 1 Fok1 will only break one strand

§  This way if there is a gene that we want to make a mutation in, ZFN can be used to break the DNA roughly where the gene is and when non-homologous end joining occurs there will be a mutation

§  Companies will design them specifically to work against the target gene

v  Transcription Activator-Like Effector Nucleases

Ø  Works similarly to ZFN

Ø  They use the same Fok1 nuclease but what makes it different is that it is fused to a customizable DNA-binding domain which comes from proteobacteria

§  The DNA binding domains are known as TALE’s

§  ‘In the wild’, proteobacteria inject their TALE’s into host plant cells by the bacteria and bind to genomic DNA to alter transcription in host cells

§  TALEs are a highly conserved 33-35 bp repeat domains

Ø  Functional TALENs work similarly to ZFNs as when the TALE’s bind to Fok1, 2 TALENs come at opposite ends and create a break in the DNA and like before will create mutations due to errors in non-homologous end joining

v  CRISPR/Cas-mediated mutagenesis

Ø  Cas9

§  Main enzyme used in this method

§  Comes from streptococcus bacteria

§  It is an endonuclease (cuts DNA)

§  Compared to Fok1 which is a single stranded cutter, Cas9 is a double stranded cutter therefore you only need one molecule to chop both strands unlike Fok1 where you need 2

§  In the wild it is part of the bacterial acquired immunity system against viruses

Ø  Cas = CRISPR-associated

Ø  Based on clustered regularly interspaced short palindromic repeats (CRISPR)

§  This is how its immune system works and remembers pathogens

Ø  This method cleaves DNA by using short RNAs

§  CRISPR-RNA (crRNA) and transactivating CRISPR-RNA (tracrRNA)

§  The RNAs bind to the complementary DNA and bind to Cas9 and creates a double stranded break

·        This system can only cut DNA upstream of PAM (Protospacer adjacent motif) sites which can vary between sites but commonly for experimental Cas9 are base pairs ‘NGG’

¨      ‘N’ can be any base pair but the 2 following must be ‘G’ and so cas9 will cut just after (2-5 base pairs after) these base pairs

·        crRNA has 2 parts, and one part will bind to complementary DNA and the other part binds to its complementary tracrRNA

·        This DNA/crRNA/tracrRNA complex brings Cas9 to its PAM site where it can cut

§  When Cas9 cleaves DNA it is not a clean break, rather it tends to be a raggedy brekja with single strand overhangs

Ø  This method is easier to replicate yourself instead of paying lots for the other methods

Ø  Studies have shown that it can create multiple mutations in embryonic stem cells and then can go on to make mutant animals

Ø  You can use this method to introduce loxP sites

v  These are 3 different techniques of genome editing

Ø  All three find ways to direct a DNA-cleaving enzyme to specific sites in any genome where you want to make mutations

Lecture 6 – Cloning Technology

v  John Gurdon (one of Martin’s lecturers) and his team got the Nobel prize for showing in xenopus frogs that transferring the nucleus of somatic cell into the oocyte cytoplasm could reprogramme the nucleus to make a clone

Ø  Shows that ‘terminal’ differentiation can be reversed because every cell in the body has the same amount of DNA therefore even taking a skin cell, it has full DNA to make a clone but they only express skin genes as the rest is turned off

Ø  Terminal differentiation is the final stage of cell differentiation, where cells permanently lose their ability to divide and take on specialized functions

v  Tracy the transgenic sheep

Ø  Made to express alpha-1-antitrypsin (AAT) in milk by putting beta-lactoglobulin promoter in front of the AAT transgene so that cells in the udders making milk express AAT

§  AAT is used therapeutically for certain lung diseases and is quite hard to make naturally

Ø  They did this many times making many sheep, but they varied in their AAT expression as you’d expect as the method would have varying results

Ø  Tracy was the sheep that produced the most drug

Ø  Tracy had babies and even they weren’t producing the same level of the AAT protein

Ø  If Tracy was cloned in the way Dolly was – using somatic cell nuclear transfer then the clone would have similar levels of AAT

v  2 sheep known as Megan & Morag (1995)

Ø  Produced by taking foetal cells that had been grown in culture, taking its nuclei out their cells and putting them into enucleated sheep oocytes 

Ø  They used TNT4 cells which are initially pluripotent but differentiate in culture

Ø  Ultimately once Megan and Morage were born, it proves that differentiated cells were totipotent if reprogrammed

v  2 sheep known as Taffy & Tweed (1995)

Ø  Fibroblast cells from epidermis of embryos of Welsh Black sheep kept in cell culture

Ø  Fused with enucleated Scottish Blackface sheep oocytes

Ø  34 reconstructed embryos transferred into 10 females, 4 got pregnant and one gave birth to Taffy & Tweed

Ø  This shows that FULLY DIFFERENTIATED EMBRYONIC CELLS REMAIN POTENTIALLY TOTIPOTENT

Ø  This is exciting cos embryonic fibroblasts are easy to maintain and mutate before making new sheep

v  Meiosis is arrested in the oocyte during ovulation therefore it had no nuclear membrane and still needs to undergo the 2nd meiotic division

Ø  It is held in the state by maturation promoting factor (MPF)

§  MPF = complex of a protein kinase cdc2 with a Ca2+-sensitive cyclin

§  High levels of MPF are required for cell division – condenses DNA, breaks down nuclear membrane

v  When the sperm enters the egg during fertilisation calcium is released, destabilising MPF making the oocyte complete meiosis

Ø  The oocyte is diploid so when the sperm meets the egg, half of the DNA gets ejected as a polar body and the other goes on to form the haploid genome that is fertilised by the sperm

v  Zygote with separate male & female pronuclei

Ø  Both pronuclei enter mitosis independently because of an increase in MPF before becoming a 2-cell diploid embryo

Ø  It is only at the 2-cell embryo stage that the cells contain a nucleus with a diploid genome

v  Somatic cell nuclear transfer

Ø  This is how they made Dolly the sheep – the cloned sheep made from adult sheep

§  Dolly made from cultured adult mammary epithelial cells and this shows that differentiated cells from adults could be reprogrammed to totipotency

Ø  The fact that before fertilisation, the oocyte is arrested due to its cytoplasm being in a high MPF state is good because it means that the donor nucleus that is inserted will break down and expose the chromatin to the oocyte cytoplasm

Ø  The donor nucleus should be in G1 or (better, not in cell cycle, G0) as once it reaches G2 there is a danger of DNA replication and it being more-than-diploid

§  To stop it from progressing through the cell cycle, the nucleus can be cultured starving it of mitogenic growth factors

Ø  Similarly for fertilisation as the sperm enters the egg, there is calcium release to destabilise the MPF and activate the egg so in somatic cell nuclear transfer the same thing needs to happen

§  To actuvate the cloned zygote artificially you can either place it in strontium or you can give it an electric shock – both methods release calcium from intracellular stores….

v  Polly the sheep (1997)

v  Cloning a mammal steps

Ø  Take somatic cells from the donor animal to be cloned

Ø  Take the chromosomes out of a recipient unfertilised oocyte

Ø  Put the nucleus of the donor cell into the enucleated oocyte

§  Direct injection or electrofusion both very efficient

Ø  ‘Activate’ the oocyte to start its development

§  Donor DNA decondenses, is ‘reprogrammed’ by cytoplasm of host, so becomes totipotent again

§  Loss of epigenetic silencing of genes – heterochromatin etc.

Ø  Transfer it to the uterus of a pseudopregnant female

Ø  Complete development as normal in utero

v  Perceived problems with cloned animals

Ø  Dolly died young of lung disease

Ø  Viability of cloned embryos very low

Ø  ‘Large offspring syndrome’ common

Ø  Respiratory and circulatory problems common

Ø  Weak immune system

Ø  Liver failure

Ø  Premature ageing (including arthritis)

Ø  Burgstaller & Brem, 2016. Aging of cloned animals: a mini review. Gerontology 63:417-425.

Ø  Why?

§  Failure of epigenetic reprogramming (some retention of DNA character
of donor cells) – this is a technical not a biological hurdle

§  Shortened telomeres - yes in sheep and goats, not in all species

§  Retention of mutations that happened during life of donor – very likely to
happen

Lecture 7 – Induced Pluripotency

v  The DNA in somatic adult cells is reprogrammable and this can be exploited for out benefit however there are ethical concerns associated with disruption of blastocysts and creation of human ES cells

v  Takahashi & Yamanaka 2006. Cell 126, 663-676 Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors

Ø  Proved that adult somatic cells can be induced to become pluripotent by forced expression of a few key transcription factors = c-Myc, Oct4, Sox2, Klf4 – induced Pluripotent Stem cells (iPS)

Ø  An alternative sources of ES-like stem cells for regeneration

Ø  Genes needed for pluripotency in ES cells and early embryos (see lecture 1) Oct4, nanog and sox2

Ø  Several genes that are commonly expressed in tumours - Stat3, E-Ras, c-Myc,
Klf4, b-catenin are required for long-term maintenance of ES cell phenotype and
rapid proliferation in culture

Ø  These scientists took 24 genes including the ones mentioned above and clones them into retroviruses and infected mouse cell in culture

Ø  Method

§  Used mice in which the coding region of the Fbx15 gene (a gene expressed in
pluripotent cells but not required for pluripotency) had been replaced by
neomycin resistance (knock-in)

§  Cultured somatic cells (skin fibroblasts) from these mice and infected them
with retroviruses expressing the 24 genes

§  Added neomycin to medium – only survivors should be the cells in which
pluripotency has been induced – iPS

§  Succeeded! Then by repeating the experiment but leaving out one or more of
the 24 genes, they found that forced expression of 10 of these genes was
enough to get pluripotency (iPS-MEF10 cells)

§  Leaving out more genes, found expression of 4 genes in somatic cells, Oct4,
Sox2, Klf4, c-Myc, was enough (iPS-MEF4 cells)

·        Nanog was dispensable as its dependent on expression of sox2 genes

Ø  Result showed that with 3 transcription factors they could still grow and differentiate but without pluripotency meaning that you need at least the 4 factors mentioned above

Ø  To test whether the iPS cells had pluripotency like ES cells, they can be implanted under the skin of an immunocompromised mouse or make them form embryoid bodies by taking away their LIF

§  The iPS cells with the 4 genes mentioned above generated teratomas proving its pluripotency whereas with iPS cells with the 3 genes showed tumours with undifferentiated cells meaning that is not pluripotent

Introduction to Induced Pluripotency

Induced pluripotency refers to the reprogramming of adult somatic cells to an embryonic stem cell-like state. The DNA in somatic adult cells is reprogrammable, which can be exploited for our benefit; however, this raises ethical concerns related to the disruption of blastocysts and the creation of human embryonic stem (ES) cells.

Cloning and Its Therapeutic Potential

Cloning via somatic cell nuclear transfer involves:

  • Taking a skin sample from a patient.

  • Isolating fibroblasts and removing the nuclei.

  • Inserting nuclei into unfertilized human oocytes to create blastocysts.

  • Extracting inner cell masses to develop embryonic stem cells for cell replacement.

Ethical consideration: The act of creating and destroying human blastocysts raises the question of when life begins.

Takahashi and Yamanaka's Breakthrough

2006: Takahashi and Yamanaka published "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors" in Cell 126, 663-676. They proved that adult somatic cells can be induced to become pluripotent through the forced expression of a few key transcription factors: c-Myc, Oct4, Sox2, and Klf4, leading to the creation of induced pluripotent stem cells (iPS). This provided an alternative source of ES-like stem cells for regeneration.

Mechanism

  • Key genes required for maintaining pluripotency in ES cells and early embryos include Oct4, Nanog, and Sox2.

  • Several genes commonly expressed in tumors, such as Stat3, E-Ras, c-Myc, Klf4, and β-catenin, are essential for long-term maintenance of the ES cell phenotype and rapid proliferation in culture.

  • Takahashi and Yamanaka initially took 24 genes associated with pluripotency and cloned them into retroviruses to infect mouse cells in culture.

Method

  • They used mice whose Fbx15 gene had been replaced with a neomycin resistance marker.

  • Cultured somatic skin fibroblasts from these mice and infected them with retroviruses expressing the 24 genes.

  • Neomycin was added to the medium; only the survivors had induced pluripotency (iPS).

  • Their experiments indicated that forced expression of 10 genes was sufficient for pluripotency (iPS-MEF10 cells), and eventually, they found that the expression of the 4 genes (Oct4, Sox2, Klf4, and c-Myc) was enough to achieve induced pluripotency (iPS-MEF4 cells). Nanog was dispensable as it depended on the expression of Sox2.

Testing Pluripotency of iPSCs

To test whether the iPSCs had pluripotency like ES cells, they could be:

  • Implanted under the skin of an immunocompromised mouse.

  • Induced to form embryoid bodies by removing LIF.

The iPSCs with the 4 key genes generated teratomas, confirming their pluripotency, while iPSCs with only 3 genes produced tumors filled with undifferentiated cells, indicating a lack of pluripotency.

Mechanisms of Pluripotency Maintenance

Several genes are crucial for maintaining pluripotency in embryonic stem cells:

  • Nanog and Sox2: Key factors in maintaining the undifferentiated state.

  • c-MYC: An oncogene linked to cellular proliferation and transcription activation.

  • Beta-catenin: Functions within signaling pathways crucial for pluripotency.

Induced Pluripotent Stem Cells (iPSCs)

iPSCs were successfully generated from mouse somatic cells and later human cells, retaining the ability to:

  • Differentiate into various cell types (verification through teratoma formation in immunocompromised mice).

  • Contribute to all tissues in developing chimeric mice when injected into embryos.

  • Show clinical potential for regenerative medicine, especially for patient-specific therapies.

Safety and Ethical Considerations

The use of c-MYC poses a risk of tumorigenesis due to its role as an oncogene. Researchers continuously explore ways to ensure safety (e.g., removing oncogenes post-reprogramming). Ethical debates continue regarding the implications of iPSCs, especially concerning potential misuse of technology.

Applications in Disease Modeling and Therapy

Sickle cell anemia model:

  • Techniques to repair beta-globin gene mutations using patient-derived iPSCs demonstrate proof-of-principle for human application.

  • Skin fibroblasts of sickle cell mice were used to create iPSCs that were then corrected genetically and successfully reintroduced into the mice.

  • Potential applications in personalized medicine, drug testing, and effective therapies for genetic disorders.

Challenges and Future Directions

Epigenetic memory in iPSCs may hinder their efficiency and differentiation capabilities:

  • Some studies found iPSCs less effective in differentiating into cell types compared to embryonic stem cells.

  • The scientific community continues to work on refining methods to enhance the quality and efficiency of iPSCs.

  • Advances in culture conditions, gene editing capabilities, and understanding of cellular mechanisms enable broader applications of iPSCs in regenerative medicine.

Conclusion

Induced pluripotency has revolutionized stem cell research and holds significant promise for future therapies. The potential to create patient-specific cells offers tremendous advantages in treating a variety of conditions, but ongoing research is needed to address the technical and ethical challenges involved.