GILESTRO - genetic screening Flashcards
Genetic Screening (targeting random genes) - type 1
- Random mutagenesis screening using chemical mutagens
* utilize alkylating agents ex. Ethyl methanesulfonate (EMS)
* adds ethyl group to O6 of guanine to create O6-ethylguanine which disrupts the WC base-pairing/ H bond with C, so G pairs with T instead, causing point mutations
Pros vs cons recessive mutation
pros:
* Easier to induce loss of function ex. by introducing early STOP codon/ cause point mutation in domains responsible for the catalytic activity
* Therefore, more common, more mutations to choose from, & have more chance of discovery
cons:
* The mutations could be lethal since both copies of the gene are modified and that mutation might not be compatible with development of the organism.
* Therefore, limited to genes that do not affect development
Offspring for mutagenesis in Drosophila
Offspring homozygous for mutation = studied for recessive mutation = results in loss of function
Offspring heterozygous for mutation = studied for dominant mutation = gain of function
* Can be a dominant negative mutant: involves a “poison” mutant peptide that
negatively interferes with the co-expressed wild-type protein, thus affecting the whole complex’s function (although only 1 component of the protein complex is mutated)
Chemical mutagenesis in Drosophila:
- Male flies eat food with EMS to induce mutations in each cell, including sperm cells
- Outcross the mutagenized males to WT females
- Generate offsprings that each have different mutations corresponding to the mutated sperm cells (F1)
- Outcross the mutated offspring individually with WT females (produce F2)
- Inbreed each set of offspring gives rise to population of flies for each new mutation (F3)
pros vs cons dominant mutation
pros:
* Easier to clone & identify the modified gene
* Therefore, more informative about the gene function
cons:
* Gain of function is more difficult since a specific mutation that results in a constitutively activated enzyme must be induced (so it does not matter if the other copy is not mutated –mutated copy can always be produced and perform the modified job)
* Therefore, less common and fewer mutations to choose from
drosophila life cycle
- Male and female mate to create a fertilized egg
- Within 24 hrs., the fertilized egg develops into a larvae
- Larval goes through 3 stages of growth that lasts about 1 week (stages differ in body size – larvae get larger and all they do is eat)
- After 1 week, larvae will feel an instinct to pupate (develop into pupa) – so larvae will climb on to a high space to create a pupa case
- within pupa case = metamorphosis to later develop into adult fruit flies
Complication of phenotype (how complicated to detect the changes in phenotype) will affect the throughput of screening
Ex. body size (can just observe from various offspring produced) vs. sleep deprivation (have to consistently keep drosophila awake) or response to pain (have to consistently disturb the drosophila)
o the help of robots can increase the throughput of phenotypes (ex. robots that repeatedly keep flies awake, repeatedly disturb flies by generating hot surfaces) and machine learning can lead better observation of change in phenotype
Transposable Elements
- fragments of DNA that can insert into new chromosomal locations and often make duplicate copies of themselves in the process, affecting the genes around the location they are inserted in
- contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material
Ex. of transposon in drosophila – P element
- consists of 4 exons
- Can be alternatively spliced in 2 isoforms - short & long depending on presence/ absence of intron 3
o presence of intron 3 shifts the reading frame to contain an earlier STOP codon, producing the short isoform – which is a repressor that will bind & inactivate the transposase itself
o splicing out of intron 3 results in full length long isoform which is an active transposase - Alternative splicing is tissue-specific
o short P element isoform is usually present in somatic cell to ensure genome stability
o long P element isoform is present in germ line cells to generate genomic variability in the germ line due to active transposase allowing mobilization of transposons - Alternative splicing is sex-specific
o P element is only present in male drosophila NOT female
Induced transposon activity:
- Can inject active transposase in male flies with P element in their genome to get active transposase activity in the germ line cells & get random insertion of transposon in random position of genome
Depending on location of transposon insertion = diff. outputs (affect gene in diff. ways)
A: In enhancer sequence
o alter the enhancer so the promotor is not recognized as efficiently, resulting in change in gene expression pattern
* B: In exon region/ 5’ UTR
o can disrupt the coding sequence, producing non-functional protein
o or if the 5’UTR is not important for the protein, can still get a functional/ partially functional protein
o P elements = likely to end up in position B
* C: In the boundary between an exon and an intron
o affect splicing of gene – mRNA not correctly spliced
* D: In intron
o nothing might happen because intron is not in the mRNA & final protein product, thus not interfering with the protein function at all
Therefore: transposon generates random mutations in multiple ways: don’t know which genes are affected & don’t know how the genes will be affected
Gene Targeting (targeting specific genes)
- Exogenous expression of a gene via transgenesis
Transgenesis by transposon
- Generate plasmid vector containing the desired gene with flanking regions that are same as a transposase flanking region
- Mix in the target organism with a helper plasmid containing the transposase sequence lacking the flanking regions – so they are not able to transpose itself, only able to transpose desired gene
- Transposase will recognize the flanking elements and think that the desired gene is one of its kind, thus performing its enzymatic activity to mobilize the gene and integrate the gene into the genome of the organism
- Back cross with the WT organism & select for transformed offspring
* Utilize transposase ability to mobilize and replicate itself to diff parts of the genome to mobilize and replicate the desired gene
* Not the same as knock in (induce expression of a gene that already exists in the organism) BUT can introduce a completely new gene by inserting an exogenic piece of DNA from scratch
Plasmid injection:
Full transposase form is always injected – so sex-specific alternative splicing does not affect the organism
* The plasmids are inject into embryo when it is just a fertilized egg, so as the infected cell duplicate, all cells will contain the modified gene – plasmids can hit all nuclei
* Some cells will also develop into germ line cells, so the progeny of injected cell will also bear the modified genome – get a stable transgenic line
* Need a marker gene to identify transformants since the process of transgenesis by transposon is not 100% efficient (depends on location of insertion)
o Use a dominant marker – a gene that creates a dominant phenotype ex. beta
galactosidase, GFP
Transgenesis by GAL4/UAS
- Fuse GAL4 under desired enhancer/ promotor (expressed in certain cells of organism)
- Fuse UAS sequence upstream of the target gene
- Fertilization of GAL4 & UAS flies will lead to controlled expression of target gene in specific cell/tissue types (that expresses the specific enhancer/ promotor) in the offspring line
- GAL4/UAS is not normally present in the organism thus their expression does not interfere with other processes in the cell
Used to:
* Study expression of a driver gene (gene under the same enhancer/ promotor as GAL4
o Ex. GAL4/UAS expressing GFP
§ Expression of GAL4 will induce UAS enhancing GFP expression
§ Observation of GFP = gene under same enhancer/ promotor as GAL4 is also expressed
* Transgenic expression of a target gene under UAS enhancer
In drosophila, it is easy to create transgene line of GAL4/UAS due to transposase activity – can randomly insert it to the genome and select mutant with the desired position of insertion
