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Behavioural Neuroscience > Genetic Tools > Flashcards

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
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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
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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)
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4
Q

Methods for obtaining transgenic animals

A
  1. Germ line modification
  2. Microinjection of DNA/gene construct into zygotes (unicellular embryos)
  3. Incorporating modified cells (embryonic stem cells) into later stage embryo
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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
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6
Q

Reverse genetics

A
  • specific gene/DNA sequence is targeted and manipulated

- from this, the function is estimated

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7
Q

Methods of DNA insertion into cell

A

-transformation=
prokaryotic cell
-transfection = eukaryotic cell

  1. Calcium precipitate
  2. Electroporation
  3. Lipofection
  4. Microinjection
  5. Viral vectors
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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
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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
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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
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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%
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12
Q

Viral vectors

A
  • modified virus is used as a carrier

- most efficient method of introducing new foreign DNA into a cell

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13
Q

Knock-out

A

-gene deleted from genome

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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
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15
Q

Knock downs

A
  • gene expression reduced

- using RNA interference techniques

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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
  1. Isolate gray mouse embryos at blastocyst stage
  2. Make cell culture from embryonic stem cells from gray fur blastocyst
  3. transfection with homologous recombinant construct
    • removal of non-transfection cells with antibiotics
  4. Injection of homologously recombined cells into white mouse blastocyte
    • creates chimeric embryo
  5. Implantation of chimeric blastocysts into pseudo pregnant white mouse
  6. 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
Q

TetOff/TetOn systems

A
  • 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
Q

Cre/loxP and Flp/FRP systems

A
  • 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
Q

Cre/loxP for gene expression

A
  • excise STOP sequence via loxP andCre

- promoter will then activate gene for transcription

28
Q

Zinc finger nucleases

A
  • 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
Q

CRISPR/Cas

A
  • 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
Q

Gene targeting

A
  • 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
Q

Transposing

A
  • 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
Q

RNA-induced gene silencing

A
  • 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
Q

Transfection using viral carrier

A
  • 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
Q

Nuclear transfer

A
  • 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

Behavioural Neuroscience (6 decks)

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