1- transgenesis Flashcards
what are transgenic or genetically modified animal lines
genome has been stably altered in all cells, including cells that form germline
germ cells are _____ cells
haploid
somatic cells
non-germ cells that do not contribute to subsequent generations
Embryonic stem cells
derived from undifferentiated cells of blastocyst ICM and can be pluripotent. they also can be maintained in culture and retain the ability to form all differentiated cells
induced pluripotent stem cells
somatic cells that is made into ES cell via reprogramming with ES cell genes
chimera
an animal containing cells from 2 different individuals
tissue specific stem cells regenerate what
specific tissues
what is transgenesis
- modification of germ cells
- permanent alteration of genome
- offspring can inherit
what is gene therapy
- modification of somatic cells
- alteration can be stable or transient
- does nothing for future generations
steps to embryo dev
- fertilized egg
- morula
- blastocyst
- early blastocyst with ICM
- embryo
what methods to produce transgenic animals
- microinjection or DNA into fertilized eggs/pronuclear injection of DNA
- gene targeting in ES cells
- nuclear transfer from genetically altered cells (cloning)
- DNA editing using site directed nucleases
does microinjection of DNA use germ or somatic cells
germ cell
does gene targeting use germ or stem cell
stem cell
does nuclear transfer from genetically altered cells use somatic or germ cells
can use both
does dna editing use germ or somatic cell
germ
what do you inject in the first method (pronuclear injection)
promoter
cDNA (coding) : AKA only exons
Poly A sequence: end recognition sequence
general process of pronuclear injection
- DNA injection into male pronucleus of fert. egg (before nucleuses merge)
- fert. egg grows into embryo
- introduce several embryos into mouse
- birth
- somatic cells are tested for presence of gene
- whichever mice have the presence of the gene are bred to test for the gene in germ cells
what are 2 ways you access the germline?
- change DNA of fert. egg
- Blastocyst and ESCs by changing/adding cells of ICM
What is a pro for pronuclear inj?
Fairly easy to perform
Can examine wide range of things
Wide species range
Cons for pronuclear inj?
-Not a lot of control
-CNVs randomly inserted in head/tail arrays
-rearrangements and deletions may occur
What is gene targeting in ES cells ?
Involves introducing a DNA construct at a precise site within the genome which allows specific mutations or deletions in the ESC allele
Then, replacement occurs by delivering constructed DNA and selecting for low homologous recombination probability
What special factor is needed for gene targeting?
Requires lines of ESCs that can be propagated in culture and also be able to integrate into a developing embryo and differentiate into germ cells
Cons of gene targeting
ONLY IN MOUSE
Pros of gene targeting
Can replace genes, knock gene out, insert gene
More control
Process of gene targeting
- Introduce a DNA fragment with the altered gene into many ES cells
- Grow to form colony
- Test for the rare colony in which the DNA fragment has replace one copy of normal gene
- Inject ES cells into early embryo after mice have mated and the embryo is retrieved
- early embryo partly formed from ESC
- Introduce early embryo into mouse
- birth
- somatic cells of offspring is tested for gene
- these mice are selected and bred to test for gene in germ line cells
What is created from gene targeting
chimera
What is nuclear transfer?
Cloning, full ESC
Process of nuclear transfer
- Take somatic cell nucleus and remove pronuclei from haploid egg cell
- inject somatic cell nucleus into the haploid egg cell
- In vitro oocyte activation/embryo culture
- Embryo transfer into animal
Disadvantages of nuclear transfer/cloning?
-Epigenetic problems - gene expression varies
-process is inefficient
Advantages to nuclear transfer/cloning?
-Germline access to virtually all mammalian species
What is gene editing?
CRISPR/Cas system
-Alters a gene rather than adding anything
What is CRISPR
clustered regularly interspaced short palindromic repeats
what is CAS
CRISPR associated nuclease
what does CRISPR-Cas 9 do?
-Alters a gene by injecting:
1. Guide RNA with homologous target sequence
2. CAS9
3. Replacement DNA
-Cas9 makes double stranded cut at target region
-Either knockout or edits the region by adding double stranded DNA that inserts into the cut
Pros of gene editing
- broad species relevance
- rapidly evolving and increases in power
- can possibly create multiple gene modifications at the same time
Con of gene editing
- less efficient for large DNA additions
In gene targeting we use _____ and _____ selection
positive and negative
Positive selection does what?
Selects for recombinants
Negative selection does what ?
ensures the homologous recombination happened
what is usually used for positive selection
antibiotic resistance
