Genetic Tools Flashcards
1
Q
History
A
- 1976: Rudolf Jaenisch introduced exogenous DNA virus into mouse embryos which started era of directed genetic alterations
- 1978 Boyer inserted human insulin into E choli to produce synthetic human insulin
- 1981: Gordon and Ruddle reported birth of mouse that had been genetically modified through microinjection of purified DNA into single cell mouse embryo
- 1996: birth of Dolly - first ever mammal cloned from adult somatic cells
2
Q
First transgenic mice
A
- Palmiter and Brinster (1980s) created important transgenic mice
1. Injecting metallotionein-rat growth hormon fusion gene into mouse embryo resulted in creating mouse double weight of unaffected control
2. Creating trangenic onco-mice by injecting metallotionein-SV40 Timor virus gene
3
Q
Advantages of transgenic animals
A
- bioreactor for synthesis of proteins
- produce human like antibodies
- model to study human disease
- study development functions of human genes
- genetically modified animals for transplantation (pig heart valves)
- transgene may speed up and reduce cost of production of animals raised for food (super salmon)
4
Q
Methods for obtaining transgenic animals
A
- Germ line modification
- Microinjection of DNA/gene construct into zygotes (unicellular embryos)
- Incorporating modified cells (embryonic stem cells) into later stage embryo
5
Q
Forward genetics
A
- examines animals with morphological phenotypes to identify abnormal gene
- altered genes are results of random mutations
- possibly due to radiation or chemical mutagens
6
Q
Reverse genetics
A
- specific gene/DNA sequence is targeted and manipulated
- from this, the function is estimated
7
Q
Methods of DNA insertion into cell
A
-transformation=
prokaryotic cell
-transfection = eukaryotic cell
- Calcium precipitate
- Electroporation
- Lipofection
- Microinjection
- Viral vectors
8
Q
Calcium precipitate
A
- DNA precipitated with calcium salts
- precipitated material is incorporated into endosome by endocytosis then released into cytoplasm
- enters nucleus
- simple method but inefficient
- most of DNA degraded in endosome
9
Q
Electroporation
A
- cells and foreign DNA put together in solution and electric current applied
- this opens pores in plasma membrane allowing entry of DNA
10
Q
Lipofection
A
- lipid micelles
- facilitates a passage of genetic material through cell membrane by formation of complexes involving cationic lipid molecules and anionic DNA
- most popular and simple lies method
- no special equipment needed
- applicable to most cell types
- less efficient than electroporation
11
Q
Microinjection
A
- DNA injected directly into nucleus
- requires specialized equipment and skilled person
- used for insertion of gene vectors into fertilized oocytes
- success rate of 4-8%
12
Q
Viral vectors
A
- modified virus is used as a carrier
- most efficient method of introducing new foreign DNA into a cell
13
Q
Knock-out
A
-gene deleted from genome
14
Q
Knock ins
A
- genes inserted into specific locus
- usually altered gene that carries point mutation
- useful for creating animal models of human diseases
15
Q
Knock downs
A
- gene expression reduced
- using RNA interference techniques
16
Q
Inserting artificial chromosomes
A
-used to create mice genetically modified with human genes
17
Q
Homologous recombination
A
- DNA repair pathway
- normally repairs double stranded DNA
- also occurs during meiosis
- can also be repaired by non-homologous end joining which fuses broken ends in more error prone fashion
- understanding this enabled development of targeted inserting a transgene into mouse genome
18
Q
Homologous recombination technique
A
- process of nucleotides exchanged between identical or similar DNA sequences
- make DNA construct we want to insert into chromosome in place of wild-type allele
- usually contains reporter gene (antibiotic resistance or green fluorescent protein)
- cells undergo homologous recombination
19
Q
Creating knock-out mouse
A
- Isolate gray mouse embryos at blastocyst stage
- Make cell culture from embryonic stem cells from gray fur blastocyst
- transfection with homologous recombinant construct
- removal of non-transfection cells with antibiotics
- Injection of homologously recombined cells into white mouse blastocyte
- creates chimeric embryo
- Implantation of chimeric blastocysts into pseudo pregnant white mouse
- Offspring with recombinant stem cells produce gray patches
20
Q
Nullizygous
A
- aka homozygous
- hemizygous= heterozygous
21
Q
Speeding up process of making knock-outs
A
- use laser beam to make hole in zona pellucida of 8-cell embryo
- introduce targeted ES cell into embryo
- generate mice than contain less than 0.1% of host contamination
- injected ES carrying homo/heterozygous mutation can be directly used for phenotypic analysis’s
- expensive method
22
Q
Ionizing radiation exposure
A
- old method of knock-out
- uses irradiating of entire enigmas and selection of mutated offspring next
23
Q
Chemical mutagenesis
A
- ENU exposure used as method of random mutagenesis
- causes base mispairing
- mutant animals bread as males to be crossed with wild type females to produce mutant offspring
-EMS more popular in studies with drosophila
24
Q
Knock-out limitations
A
- 15% of knockouts are lethal
- function of mice gene homologous may be different from human gene
- some knock-out effects use other methods to fill gap
- knockouts hard to reproduce
- mouse strain used for ES cell knock outs physiologically and behaviourally distinct from other mouse lines
25
TetOff/TetOn systems
- method using tetracycline-resistance operon of E. coli
- presence of antibiotic activated the protein that pumps the drug out of the cell
- TetOff: expression of target gene is suppressed by tetracyline and starts when supplying an antibiotic is stopped
- TetOn: when doxycycline present the gene is being expressed/repressed in tissue
- advantage: expression of gene can be switched on and off in chosen time
- disadvantage of TetOff is that animal has to be kept on antibiotic for a long time
26
Cre/loxP and Flp/FRP systems
- cre and Flp are bacteriophage and yeast recombinases which recognize loxP and FRT sequences
- DNA fragment between two loxP will be cut out and strands rejoined
27
Cre/loxP for gene expression
- excise STOP sequence via loxP andCre
| - promoter will then activate gene for transcription
28
Zinc finger nucleases
- ZFN are designed restriction enzymes
- can be engineered to target DNA, and produce a double strand break
- will either be correctly repaired by homologous recombination or fixed by NHEJ
- gene function may be partially/entirely disrupted
29
CRISPR/Cas
- easy to customize because site selection for cleavage is guided by short RNA sequences
- disadvantage
- obtain off target effects
- hard to detect whether mutated conventionally or genetically engineered
30
Gene targeting
- allows reliable identification, monitoring, and manipulation of neurons
- using light-induible transgenes allows us to photostimulate or photoinhibit neurons with high spatial and temporal resolution as well as in a cell specific manner
31
Transposing
- prices of DNA with ability to change positions within genome
- 2 types: copy and paste, cut and paste
- copy and past retro-transposing are mobilized by transcribing RNA copy, which then becomes reverse transcribed
- integrated elsewhere into genome
- cut and paste elements are transposed by direct excision from DNA and inserted elsewhere
- sleeping beauty transposing
32
RNA-induced gene silencing
- all silencing have short length
- small interfering RNA (siRNA)
- microRNA (miRNA)
- Piwi-interacting RNA (piRNA)
- mediate RNA cleavage but also regulate variety of biological processes
- disadvantages
- frequently observed off-target
- over expression of shRNA can competitively inhibit intracellular processing of miRNA which could cause toxic side effects
33
Transfection using viral carrier
- highest success rate but difficult to design
- minimize number of viral genes present in construct (plasmid)
- main aims are to create replication-deficient virus units and to prevent recombination mediated formation of so-called replicative competent retrovirus
- safe alternative for viral vector is using transposing
34
Nuclear transfer
- enucleation of host oocyte
- either nuclear or donor cell is fused with enucleated oocyte
- donor nuclei both obtained from either stem cells or differentiated adult cells
- somatic cell-derived nucleus exhibits cellular senescence as consequence of telomere shortening
