Lecture 21 - Genetic approaches, verifying mutant genes, targeted mutagenesis

What is forward genetics

The process of going from phenotype to determine the gene

what are the steps in forward genetics

1. Identify a variant phenotype which can be done by inducing mutations with chemicals, x-ray, neutrons

2. Make a cross between strains (backcross and complementation cross)

3. Careful analysis of alterations in cellular processes (biochemical pathway, physiological, cell biology)

4. Gene mapping and them gene cloning

What are the pros and cons of forward genetics

Pros:

1. Don’t need to have a sequenced genome

2. Can identify genes with no known predicted function

3. Garanteed results since you know there is a final effect

Cons:

1. May be difficult to identify if the mutation is complicated

2. Redundancy from genes with overlapping function would make it hard for phenotypic screening

What is reverse genetics

Reverse genetics is the type of process where you have the gene and you want to establish the phenotype it is linked to. To investigate you must make a mutation in the gene and see what may occur

What are the pros and cons of reverse genetics

Pros:

1. Not limited to phenotypes that can only be screened for when you have a large population of mutagenics

2. Knockout many redundant genes to avoid redundancy

Cons:

1. May not get any phenotype after all the work

2. hard to know out genes in non-model organism

What are the three methods for identifying and verifying mutant genes

1. Transgene insertion mutation

2. Positional cloning to test point mutations

3. Genmoe sequencing to test point mutations

Elaborate on transgene insertion mutation

To verify a transgene insertion mutation there could be the use of inverse PCR. In this method the researcher must chose a RE that is outside the transgene region and one that is inside the transgene region. Then after the restriction enzyme cuts a ligase enzyme will come in and ligate these strands to be a circular piece of DNA. Once circular you will need to design primers for PCR and PCR amplify the product/ Afterwards you will sequence this DNA and verify that the sequence of your transgene is found in this region

Elaborate on point mutation: positional cloning verfication

Essentially what is done is you need to map the area by perfoming back crosses and using genetic markers. Then you use a genome assemble to establish the candidate gene. Then you can sequence the exons of this gene and compare to verify if a point mutation has occured

Elaborate on point mutation verification that uses genome sequencing

You need to sequence the genome of the mutant strains and compare it to the genome sequence of different strains with mutation affecting same gene

When you identify where a mutation is how do you verify that your conclusion is correct

There are two ways you can go about this

1. You can rescue mutant phenotype with transgene containing wild-type gene copy

2. You can mutagenize a wild-type strain by gene tragetting or CRISPR to test if the same mutant phenotype will result

what is targetted mutagenesis

enables scientist to change specific genes in virtually any way desired

What are the steps to gene targetting

1. Mutagenize the gene in vitro (in the lab)

2. Then you put the mutant gene in the cell

3. Than a rare homologous recombination replaces normal gene with mutant gene

   • Only occurs when there is crossover between homologous regions on between the two DNAs

Does homologous recombination work well in all species

It works well in yeast but doesn’t work well in plants

How do you make knockout mice

1. You replace a functional allele with an amorphic one (non functional one)

2. Mutant DNA construct is taken up by embryonic stem cells made from agouti mouse blastocytes

3. Homologous recombination, allows for created gene to be inserted into ES cells

4. Then you will plate the cells on a new plate for selection of the organims that took up the DNA (the ones that have the drug resistance)

5. Then you inject it into a different mice’s blastocytes

6. The blastocyte is the implanted into the uterus of a foster mother mouse

7. The mice child with be chimera (have 2 cells from different genetic lines) and will mate with another mice to have a heterozygous one

What is special about embryonic stem cells

they are undifferientiated cells from blastocyst of embryos they can grow and divide in culture and are totipotent and can become any type of cell

How do you perform a knockin

1. You design your construct that has ends that match that of the genomic DNA to allow for homologous recombination, it must also contain a drug resistance. They contain loxP sites

2. Then the embryonic stem cells will be plated to check if they gained the resistance

3. Then the loxP sites will be recognized by Cre which will allow for the drug resistance to be removed

4. Final product is the wildtype exons are replaced with the mutant ones

Does loxP interfere with splicing

no it doesn’t