Lecture 21 Modern approaches to transgenesis

Question 1

disadvantages of generation of transgenic mice from ES cell modification via homologous recombination

Answer 1

laborious
time consuming
have to breed generations of mice, use different strains, pseudopregnant mice…

Question 2

methods to try target genome sequences directly and more efficient?

Answer 2

-zinc finger nucleases (ZFN)
-transcription activator-like effector nuclease (TALEN)
-CRISPR/Cas9 systems

Question 3

cas9 gRNA mediated cleavage?

Answer 3

Make up an RNP complex
-cas9 endonuclease
-+ a guide RNA (gRNA)

Question 4

gRNA properties

Answer 4

should contain specific 20nt protospacer (the target sequence) which is designed to target upstream of Protospacer adjacent motif (PAM “NGG” N= any base nucleotide)

Question 5

Cas9 properties

Answer 5

Endonuclease - recognises the PAM site upstream of gRNA target site and makes double-stranded breaks 3 nt upstream of PAM

Question 6

3 scenarios after Cas9 and gRNA directed DNA cleavage

Answer 6

1. Repaired by non-homologous end joining (NHEJ)
   used for gene knockouts
   (NHEJ can remove nucelotides in its repair process)

or

2. Repaired by HDR (homology directed recombination, RecBCD -> RuvABC pathway):
   use short oligonucleotides or DNA vectors with short homology arms that flank the cleavage site
   used to introduce specific mutations, reporter (GFP…), or loxP sites (IDK) etc

OR

3. Two gRNA sites can be chosen to delete large region of genome (i guess its then repaired by NHEJ)

Question 7

genome editing with CRISPR/cas9?

Answer 7

Cas9 and gRNA injected into nucleus
repair in vivo using HR allows rapid generation of transgenic animals -introducing the new sequence into the site

multiple sites can be targeted in a single experiment

Question 8

off target effects of Crispr/cas9

Answer 8

cas9/guideRNA cleavage is not absolutely specific

need to choose target sequences within genes with minimal homology to other genes/regulatory regions

Question 9

mutant cas9 to reduce off target effects?

Answer 9

mutant cas9 nuclease “nickase” (cas9D10A) cleaves only one of the DNA strands

only a ssBreak

use 2 gRNAs specific for opposite strands of the DNA along with cas9D10A generates an overhang

NHEJ and HDR can be used to knockout/introduce respectively

Question 10

Conditional genetics uses?

Answer 10

-Conditional deletion of a gene
>avouding early lethality of animal from mutation so effect can be analysed
>analysing role of gene in a defined cell type (cell specific inactivation)
>analysing the role of a gene at a certain developmental stage (inducible insctivation)

-permanently tag cells with a reporter gene to determine their fate

-to activate a mutation e.g. oncogene in a certain cell type

Question 11

core of conditional genetics?

Answer 11

Site specific recombination (SSR)

can be driven in vivo by expressing Cre-recombinase from a transgene

loxP sites (SSR target sites) are previously inserted by gene targeting to flank a critical sequence (using cas9, or ES cell targeting but cas9 is better)

ssr of these head to tail lox9 sites excises the sequence from the genome

Question 12

inducible deletion of genes

Answer 12

sometimes - gene only required at a certain stage of development or in adult animal
hence inducible deletor may be used

Question 13

inducible deletor: Cre-ERT2 constructs

Answer 13

used to generate deletor in mice

Cre (recombinase?) fused to mutant estrogen ligand binding receptor domain (ERT2)
ERT2 responds to synthetic estrogen analogue - Tamoxifen

ERT2 keeps the Cre recombinase in the cytoplasm until tamoxifen is added
causes Cre-ERT2 to move to nucleus and cause recombination

Question 14

inducible deletor/comditional genetics Small intestine self renewal example

Answer 14

trying to identify stem cells in intestinal crypt that renewed intestine cells
do they exist as stem cells?

specific population of cells at bottom of crypt expressing Lgr5, other cell types that dont
could it be Lgr5 in stem cells?

engineered Lgr5 mutant mouse - GFP reporter knocked into Lgr5 expression
expressed at bottom of crypt

this mouse was engineered so that:
Lgr5 promoter not only drove GFP expression but also Cre-ERT2 expression (through IRES to make multicistronic transcipt)
allows knowledge of where cre-ERT2 is expressed

had another LacZ transgene with a constitutive promoter that is expressed in all cells in the animal
has a repressor (stop cassette) of lacZ activation upstream of the LacZ sequence with lox9 sites flanking it

injection of tamoxifen activates Cre-ERT2 which excises stop cassette (through SSR) - allows LacZ expression
since Cre-ERT2 controlled by Lgr5 promoter - will only have Lacz expression in cells that express Lgr5

even when expression of Lgr5 is down regulated in daughter cells of Lgr5+ cell - LacZ expression will remain (constitutive promoter) so can see what cells are derived from the Lgr5+ cell line

injection of tamoxifen -> can then trace what cells come from stem cells (even when Lgr5 expression has stopped)

Question 15

conclusion of small intestine self renewal experiment

Answer 15

the Lgr5+ cells are the stem cells at the bottom of the crypt (as it is shown that they give rise to other cells in small intestine from LacZ)

GFP expression shows that the Lgr5+ stem cells exist only in the crypt and their LacZ expressing (but GFP non-expressing) daughters move out

Question 16

conditional genetics - deletion of oncogene

Answer 16

the same Lgr5-creERT2 mouse can be used to delete APC tumour suppressor gene specifically in gut stem cells

(creERT2 only expressed in gut stem cells as it is under control of Lgr5 promoter)

-deletion of APC gene
-activation of Beta-catenin (downstream of APC control)
-formation of microadenomas from the transformed stem cells

deletion of APC tumour suppressor gene in gut non stem cells using Ah-Cre deletor does not drive adenoma formation
only deletion in stem cells drives tumour formation

Question 17

mutated Cas9 to activate or repress genes of interest?

Answer 17

dCas9 = “dead” version of Cas9
(cannot cleave DNA so cannot mutate )

can be fused either with activator (VP64) or repressor (KRAB) of target gene next to gRNA target site

dCas9

Question 18

reprogramming of cells - somatic cell nuclear transfer (cloning)

Answer 18

reprogramming of genome
a donor somatic cell can be genetically modified in culture and placed under the zona pelucida of an enucleated oocyte

fusion of membranes of donor and recipient cell and activation of embryo development is initiated by electrical pulses

Question 19

how can somatic cell nuclear transfer be used? (SCNT)

Answer 19

-reproductive cloning - generating an animal which contains a COPY of the genome of another animal (e,g, dolly the sheep)

-therapeutic cloning - generating ES cells from a cloned embryo with intention to generate cell types in vivo for regenerative medecine

Question 20

downside of SCNT (somatic cell nuclear transfer)

Answer 20

reprogramming is never achieved with same success as fertilisation

main reason of low survival - abnormal epigenetic regulation

fertilised oocyte undergoes all necessary epigenetic changes to become blastocyst

the SCNT oocyte only undergoes partial reprogramming (genome in somatic cell already has epigenetic structure)
leads to much increased cell death

Question 21

epigenetics in reprogramming

Answer 21

ingeritance which is independent of DNA sequence
mediated by heritable but reversible modifications to the DNA
e.g. methylation of DNA, or some heritable change to chromatin (e.g. histone acetylation/methylation)

Question 22

pronuclear transfer between fertilised mouse eggs

Answer 22

oocyte and sperm pronuclei present un oocyte
remove sperm pronucleus

-then add egg pronucleus - gives abnormal fetus

-but if another sperm pronucleus is added - normal foetus

maternal and paternal pronucleus both required for normal development (due to imprinting differences)

Question 23

parent of origin imprinting?

Answer 23

-some genes are expressed/repressed depending on their parental origin resulting in monoallelic uniparental expression

-imprinting of one of the two alleles occurs exclusively during gametogenesis

-some genes are imprinted during male and others during female gametogenesis

-these imprints persist throughout the zygote and into the somatic cells of foetus and adult

imprints in that individual are then erased and re-established according to the sex of the embryo

Question 24

imprinted regions properties?

Answer 24

imprinted genes often clustered in certain regions of chromosomes

get clusters of maternally and paternally expressed genes

Question 25

revealing imprinted gene by mutation

Answer 25

one gene imprinted in maternal female gamete - wont be expressed
same gene not imprinted in paternal male gamete - will be expressed in embryo

delete in paternal but not in maternal
dont get expression in the embryo as paternal mutated and maternal imprinted to not be expressed

delete same locus in maternal but not paternal - expressed as normal in embryo

Question 26

imprinting effects:

Answer 26

currently 80 imprinted genes identified

major effects on foetal growth
-maternally expressed genes suppress while paternal ones enhance

so defects can lead to disease and cancer

Question 27

mechanism of imprinting:

Answer 27

DNA methylation major mechanism
differentially methylated regions (DMR) identifies within or close to several imprinted genes

other epigenetic modifications: histone acetylation and regulatiojn of ncRNA is also implicated

Question 28

reprogramming of differentiated cells into pluripotent cells

Answer 28

-identification of 24 genes differentially expressed in ES cells compared to differentiated cells

-transduction of embryonic fibroblasts with 24 retroviruses containing these genes

-combination of OCT4, SOX2, KFL4, c-MYC genes sufficient to generate induced pluripotent cells (iPS cells) from mouse fibroblasts

can introduce different factors to cause them to differentiate into other cell types

Question 29

theraputic use of iPS cells?

Answer 29

treating disease
mouse has sickle cell anaemia
collect skin cells
reprogram into ES cell like iPS cells (the 4 retroviruses from earlier)
get iPS cells genetically identical to sick mouse
correct the mutation causing the SC anaemia - genetically correct the iPS cells

then differentiate them into blood stem cells and transplant them into sick mouse

Question 30

potential problems with therapeutic iPS

Answer 30

-remaining undifferentiated cells can form tumours

-cell types differentiated in culture may not be fully finctional

-integration of cells and establishment of proper contact with other cells in tissues may be problematic