Genet 302 lec 21 & 22
What is random mutagenesis used for?
To generate random mutations and then isolate rare offspring with the desired phenotype
What is the classic workflow of random mutagenesis?
Expose many organisms to a mutagen, then screen offspring for the mutant phenotype
What mutagen was used in the Neurospora example?
X rays
What phenotype was isolated in the Neurospora example?
Arginine− auxotrophs
What chemical is commonly used in mice to make random base-pair substitutions?
ENU
What does ENU stand for?
N-ethyl-N-nitrosourea
What kind of mutation does ENU mainly cause in this lecture?
Base-pair substitutions
What base change example was given for ENU?
AT to TA
Was Dmdmdx-2Cv random or targeted? what is this example?
Random generated mut in dystrophin gene on MMUX
Where is the Dystrophin gene in mice?
On the X chromosome
What kind of mutation was shown for Dmdmdx-2Cv?
A splice-site mutation
Where was the splice-site mutation in Dmdmdx-2Cv if the mut a--> T was shown 2 away from the start of exon 43.
Intron42 -2 A --> T
What kind of chromosome mutations are commonly produced by gamma radiation?
Chromosome rearrangements
How does gamma radiation generate chromosome rearrangements?
It causes random double-strand breaks that are improperly repaired by NHEJ- wrong ends connected
What chromosome rearrangement example was shown after gamma radiation?
Reciprocal translocation
What mouse strain was used as a Down syndrome model in this lecture? how did this come about?
Ts65Dn. reciprocal translocation between MMU16 and MMU17
What is targeted mutagenesis used for?
To make a specific mutation in a specific gene
What was the major pre-2014 method for targeted mutagenesis in mice?
ES cells
What is the major post-2014 method for targeted mutagenesis in mice?
CRISPR
What method is used to add a gene to mice, both pre-2014 and post-2014?
Pronuclear injection
What type of mutagenesis was Dmdtm1MOK in the gene dystrophin.
targeted mutagenesis. deletion of exon 52.
What is the key summary of the lecture 21 table for random mutagenesis before 2014?
ENU and gamma rays were used
What is pronuclear injection used for?
To add a gene and make a transgenic mouse
What simple example of a transgene was used in lecture 22?
Jellyfish GFP
What two main parts are needed in the basic DNA construct for pronuclear injection?
A promoter (ex. chicken B-actin gene- active in all cells) and a cDNA of the gene of interest (ex. jellyfish GFP gene)
Why is a cDNA used instead of genomic DNA in these constructs?
cDNA contains the coding sequence without introns
What does a cDNA contain from a gene?
Exons from the mature mRNA, not promoter or introns
What is step 1 of pronuclear injection?
In vitro fertilization to make zygotes
What is step 2 of pronuclear injection?
Inject DNA (plasmid with the gene) into the sperm pronucleus at G1-larger/easier target.
Why is the sperm pronucleus injected? how?
It is visible and accessible in the zygote. using needle and suction pippete
What happens after DNA is injected into the pronucleus?
The transgene integrates randomly into a chromosome. dna repair enzymes dont like dna floating, so it integrates it into the chromosome
How does the injected DNA integrate into the genome?
By DNA repair enzymes causing random integration
What is step 4 of pronuclear injection?
Make a pseudopregnant female by mating a female with a sterile male- will reject other wise
Why is a pseudopregnant female needed?
To receive and carry the transplanted embryos
What is step 5 of pronuclear injection?
Transplant the embryos into the pseudopregnant female
What is step 6 of pronuclear injection?
Obtain offspring and screen for transgenic mice
What proportion of offspring may carry the transgene in a good experiment with a small transgene? would the gene be hemizygous or heterzygous?
Up to about 20%. hemizygous bc only half of the chromosomes would have it and the other half would have no allele at all not a different allele like heterozygous
What is the main limitation of pronuclear injection?
The transgene inserts randomly
What does tTA stand for in the tTA/TRE system?
Tetracycline-controlled transactivator
What is TRE in the tTA/TRE system?
The DNA regulatory sequence that tTA binds to
What happens when tTA binds TRE?
It activates transcription of the downstream gene
What kind of protein is tTA functionally?
A positive transcription factor
Why is the tTA/TRE system useful?
It gives controlled expression only where the promoter driving tTA is active
Why was a simple heart-specific promoter directly driving CECR1 considered a bad approach?
It could cause problems like lethality, sterility, or unwanted effects in other tissues/stages
What is in the tTA mouse strain?
A tissue-specific promoter driving tTA cDNA
What is in the TRE-YFG mouse strain?
TRE upstream of the cDNA for your favorite gene
What are the two separate mouse strains used in the tTA/TRE system?
A tTA mouse strain and a TRE-YFG mouse strain
What happens when you cross a tTA mouse with a TRE-YFG mouse?
Double-transgenic offspring express the target gene where the tTA promoter is active
What promoter was used in the CECR1 example to drive tTA in the heart?
Alpha-MHC
What did the TRE-YFG strain contain in the CECR1 example?
A bidirectional TRE promoter, human CECR1 cDNA, and E. coli LacZ cDNA
Why was LacZ included in the CECR1 experiment?
As a reporter to show where the transgene is being expressed
How can LacZ activity be detected?
With X-Gal staining
What does beta-gal do to X-Gal?
It converts it into a blue product
What must a labelled diagram for fluorescent green ears include at minimum?
A tTA strain, a TRE-GFP strain, pronuclear injection steps to make each strain, a cross between the strains, and GFP expression in ears
How would you design the tTA strain for green ears?
Use pronuclear injection to make a mouse with an ear-specific promoter driving tTA
How would you design the TRE responder strain for green ears?
Use pronuclear injection to make a mouse with TRE upstream of GFP cDNA
What do you do after making the two separate strains?
Cross the ear-specific tTA strain with the TRE-GFP strain
Which offspring will have fluorescent green ears?
Offspring that inherit both the ear-specific tTA transgene and the TRE-GFP transgene
Why will fluorescence be limited to the ears?
Because tTA is produced only in ear tissue, so TRE activates GFP only there
What would the labelled construct for the first strain look like?
Ear-specific promoter -> tTA cDNA
What would the labelled construct for the second strain look like?
TRE -> GFP cDNA
What key pronuclear injection steps should be shown in the diagram for each construct?
Make zygotes, inject DNA into sperm pronucleus, random integration, transfer embryos to pseudopregnant female, obtain transgenic offspring
What final genetic cross should be shown in the diagram?
tg(ear-specific-tTA) × tg(TRE-GFP)
What final genotype should be shown for the glowing mice?
tg(ear-specific-tTA)/+ ; tg(TRE-GFP)/+
What final phenotype should be shown for the glowing mice?
Green fluorescent ears
Why is the tTA/TRE system better than just putting GFP under an ear-specific promoter in one construct for this type of question?
It gives a modular two-strain system where expression can be controlled by combining driver and responder lines