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Lecture 23

BIOL 5060 Lecture 23: 4-25_RecDNATech_Hoffman

5 Pages
97 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
23

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4/25_RecDNATech_Hoffman 4/25/2017 3:29:00 PM
Information ab the promoter
Must be a promoter in the host cell to drive expression of insert
gene
Experiment: put something in to remove activity
o Example: in yeast we put in a marker in a way to make it
recombine and delete one copy of the gene
o RNAi= RNA interference
o dsRNA- there was a sense and an antisense promoter working
in both directions to produce complementary RNAwhich
would feed into the RNAi mechanism
o dsRNA (RNAi) RITS complex with siRNA in it that can
basepair with a transcript…
if bp is 100% there will be mRNA cleavage
o used as a methodology, going after complete destruction of
RNA
How to make dsRNA?
1) Take chunk of GOI and clone it into a vector with bacteriophage
promoters flanking it in vitro or mix Feed dsRNA into worms//
inject into cell
2) shRNA= short hairpin RNA- base pair in a single strand to form a
ds hairpin (crick to Watson)
o with a single RNA molecule, we made a dsRNAmore
appropriate if we are trying to do a stable transfection of
somethingappropriate for both transient and stable
transfections
o cells receive the clone (not the RNA) and they make the RNA
Considering mouse development…
We have an egg and a sperm that fertilize to form a zygote, which
sporulate to form a bastula mouse
If the transgene is introduced in the beginning (around
fermentation), the entire organism will be transgenic//
heterozygous transgenic mouse
If the transgene is introduced somewhere after fertilization, during
sporulation, not the whole mouse will be transgenic mosaic
mouse with only some transgenic cells
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If we want to assess the impact of the transgene, we need to think
about genetic types of mice
o Mosaic- some cells affected
Affected cells are heterozygous
o Heterozygous mouse- all cells are heterozygous
o Homozygous- all cells carry two copies of transgene
o Mosaic animals have to be bread with “normal” animals look
for transgene in progeny
Do PCR for the presence of the transgene
Heterozygous ES transgene is detected by PCR
How to introduce transgene
Injection into egg or zygote
Viral infection of blastula
Introduction by some mechanism into embryonic stem cells ES
cells
o Stem cells= mitotically active- pluripotent or omnipotent
meaning they can become any of a number of types of cells
Old school gene knockout
Mice are much more likely to have a gene with many exons (as
opposed to budding yeast- almost no genes have introns)
Identify a portion of the coding region that is essential for function
Exons for catalytic domain of enzyme
We have…
o Neo flanked by regions A and B
Neo confers G418 resistance
o and regions A and B flanking a few exons in the mouse
o how will we know if we have a homologous recombination
event?
We can do internal and flanking PCR or just flanking
PCR to screen
See if the size of the gene is different to suggest a
recombination event
OR we can have HSV-TK (herpes simplex virus) stuck
on the end of the neo flanked by A and B
It was found the HSV-TK is counterselectable on
medium that contains ganciclovir
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find more resources at oneclass.com

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Description
4/25_RecDNATech_Hoffman 4/25/2017 3:29:00 PM Information ab the promoter • Must be a promoter in the host cell to drive expression of insert gene • Experiment: put something in to remove activity o Example: in yeast we put in a marker in a way to make it recombine and delete one copy of the gene o RNAi= RNA interference o dsRNA- there was a sense and an antisense promoter working in both directions to produce complementary RNA—which would feed into the RNAi mechanism o dsRNA (RNAi) RITS complex with siRNA in it that can basepair with a transcript… ▪ if bp is 100% there will be mRNA cleavage o used as a methodology, going after complete destruction of RNA How to make dsRNA? • 1) Take chunk of GOI and clone it into a vector with bacteriophage promoters flanking it in vitro or mix Feed dsRNA into worms// inject into cell • 2) shRNA= short hairpin RNA- base pair in a single strand to form a ds hairpin (crick to Watson) o with a single RNA molecule, we made a dsRNA—more appropriate if we are trying to do a stable transfection of something—appropriate for both transient and stable transfections o cells receive the clone (not the RNA) and they make the RNA Considering mouse development… • We have an egg and a sperm that fertilize to form a zygote, which sporulate to form a bastula mouse • If the transgene is introduced in the beginning (around fermentation), the entire organism will be transgenic// heterozygous transgenic mouse • If the transgene is introduced somewhere after fertilization, during sporulation, not the whole mouse will be transgenic mosaic mouse with only some transgenic cells • If we want to assess the impact of the transgene, we need to think about genetic types of mice o Mosaic- some cells affected ▪ Affected cells are heterozygous o Heterozygous mouse- all cells are heterozygous o Homozygous- all cells carry two copies of transgene o Mosaic animals have to be bread with “normal” animals look for transgene in progeny ▪ Do PCR for the presence of the transgene ▪ Hete
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