Lecture 10 Flashcards
clone
genetically identical copy of an organism
cloning
procedures to create exact replica of a cell, tissue or organism
Natural clone
identical twins
why genetically clone?
- improved productivity/welfare
-disease resistance - reduce environmental footprint
- preserve endangered wild species
- animals as bioreactors - biomedicine/organs
what is a blastomere
cells formed through early division of a zygote
how do blastomeres form
through cleavage divisions
blastomeres are formed at what stage
2 -4cell stage
what can each blastomere develop into
an individual organism; one cell type
how many cell types are present at the blastocyst stage
2 cell types
describe the process of embryo twinning
- single cell zygote has undergone first cell division
- then the two zygotes are separated
- embryo is transferred to an unrelated surrogate mother
- sheep can give birth to identical twins
what are the pros and cons of embryo twinning
it mimics natural process but only limited number of clones can be produced
who is dolly the sheep
cloned sheep produced by somatic cell nuclear transfer
describe the process of SCNT
1a. remove nucleus of oocyte from cytoplasmic donor (enucleation)
1b. obtain donor nucleus from nucleus donor
2. introduce donor nucleus into enucleated oocyte
(nucleus of oocyte is replaced by nucleus of somatic cell)
(DNA of somatic cell is reprogrammed to pluripotent stem cell state)
3. transfer blastocyst to surrogate
(blastocyst develops into fetus in surrogate and a clone of the nucleus donor is born)
describe the general timeline of animal cloning (earliest to latest)
- sheep: embryo splitting
- cattle: embryo splitting
- sheep: embryo cloning
- cattle: embryo cloning
- sheep fetal and adult SCNT
- cattle: fetal and adult SCNT
- goat, pig
8.cat - horse
- dog
- camel
how can you clone for conservation
interspecies somatic nuclear cell transfer
what is an ex. of interpecies somatic nuclear transfer being used
domestic dog oocytes being used to clone coyotes - hybrid embryo
what are some potential problems of interspecies somatic nuclear cell transfer
- nuclear DNA of cloned species, mitochondrial DNA from surrogate mother express genes necessary for energy production –> if there is no fit, developmental problems
- embryo tissue/ placenta is recognized as foreign by immune system of surrogate mother
define reprogramming
the process of resetting adult nuclear DNA to the early development/pluripotent pattern of gene expression
(aka bringing differentiated somatic cell back to stem cell state)
why do problems sometimes occur during SCNT
Reprogramming is problematic!
- switching on and off gene expression: errors in reprogramming may lead to abnormalities in gene expression in cloned animals and affect the health and longevity of the animal
- epigenetic changes:, DNA methylation, histone modifications, imprinting, X-chromosome inactivation, chromatin remodelling
…. can lead to: pregnancy losss, congenital abnormalities and postnatal mortality
what is cloning
generating genetically identical copies of cells, tissues and organisms
what is embryo twinning
microsurgical dissection of blastomeres at an early embryonal stage (2-4 cell stage); each blastomere can develop into an individual animal in surrogate mother
(aka. separate blastomeres –> each blastomere develops into a separate blastocyst)
describe somatic cell nuclear transfer
nucleus of donor cell is introduced into enucleated oocyst, then embryo transfer to surrogate
where can the donor nuclei be obtained from for SCNT
donor nuclei can be obtained from fully differentiated cells (ex. fibroblasts)
which SCNT nuclei is more efficient when…
nuclei from early embryonic stages
SCNT was first shown in…
sheep
which part of SCNT is problematic
reprogramming- the reset to pluripotent state of DNA
what are GMOs/ transgenic animals
organisms whose genome has been altered using genetic engineering methods
how are GMOs done
through deleting genes, introducing new genes and replacing gene order
what are some goals of transgenic animals/ GMOs
- improve/ enrich animal products
- generate disease models
- animals as bioreactors - production of recombinant relevant proteins (ex . spider goats)
what opened the door for efficient creation of large transgenic animal species
SCNT
what is a transgene
recombinant DNA introduced into an organism, transgene can then be introduced into cultured cells –> becomes the nuclei of selected transgenic cells and used for SCNT
what are the steps for using SCNT for creating transgenic large animals
1a. create the transgenic cell: transfect somatic cells with transgene
1b. get oocyte and remove chromosomes for ennucleated oocyte to insert transgenic nuclei)
2. perform SCNT with nuclei of transgenic cells: ( first transgenic cells grow in vitro culture in selection, then expanded cells get transferred into ooplasm
3. embryo is transferred into recipient animal and then there is delivery of cloned calf (transgenic animal)
what are the pros of transgenics
- somatic cells can be cultured and manipulated ex vivo by introducing transgenes
- transgenes/ recombo. DNA is incorporated into genome of cultured somatic cells
- nuclei of manipulated somatic cells for SCNT
what are the downsides of using transgenes*
- transgene integrates randomly into genome - no control where and how many copies
- could have integration of transgene into sliced gene regions –> no expression of transgene
- expression to high if multiple copies are integrated –> high number of transgenes may be toxic
genome/ gene editing
the ability to make changes at a specific site in the genome of an organism (deletion, replacement, editing/correcting DNA sequences)
what is needed to achieve genome/ gene editing
- nuclease: introduce double strand breaks into DNA
- mechanism/component to direct the nuclease to the specific location in the genome
- cellular DNA repair mechanisms that are activated in response to DS DNA breaks
DNA double strand breaks (DSB) are what form of DNA damage
the most serious kind!
how are dDSBs formed
through endogenous processes (ex. DNA replication) or exogenous agents (UV radiation, certain chemicals)wha
what happens when there is failure to repair DSBs
cell death
what are 2 mechanisms to repair DSBs
- non homologous end joining
- homology directed recombination
how does Non-homologous end joining work
- proteins recognize the breakage (direct recognition)
- polymerase and lygase recognize and join DNA (processing)
- 2 ends are joined together`
what are the downsides of NHEJ
process is highly error prone
- can introduce indels into DNA
- frameshift/gene knockout
What is homology directed recombination (HDR) used for
- used to insert, delete or replace DNA sequences
describe the repair template in HDR
linear DNA, homologous regions flanking the target DNA
name 4 genome/gene editing tools on a scale from least to most feasible
meganucleases, zinc finger nucleases, TALEN, CRISPR/Cas9
what are the most common genome/gene editing tools
Zinc finger nucleases, TALEN, now CRISPR/Cas9
what is the nuclease used in zinc finger nucleases
FokI
what is the DNA recognition component for ZFN
zinc finger motif (protein)
describe zinc finger motifs
consist of 30 aa, recognizes triplet (3 nucleotides)
typically, how many nucleotides are targetted and how many motifs are used to guide FokI to specific location
typically 9-18 NTs are targeted (3-6 motifs)
what is the role of Zinc finger motifs
recognize sequence and where to put the DS DNA
what are the cons of ZFNs
inflexible: relies on what motifs are available
- want to avoid off target effects
what does TALEN stand for
transcription activator-like effector nucleases
