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Texto Diapositivas Martín
Texto Diapositivas Martín
Texto Diapositivas Martín
Diapo 7.- To do so, we searched in the literature for genes induced by the presence of FAs, to
take their promoters and put the right before a Cre sequence. Mammalians FAs metabolism is
regulated by two transcription factors, PPAR and RXR, that respond to the presence of FAs by
their co-activation. This had a great inconvenient; they can interact with p38alpha signalling
pathway. However, we noticed there is a homologous system in yeast that wouldnt interfere
with the mammalian metabolism, being useful for our aim. These proteins are Oaf1p and Pip2.
Both in presence of FAs they bind to a promoter called Oleate response element and start
the transcription of proteins involved in FAs metabolism.
Diapo 8.- According to this, we thought about the following system. Using the Ins1 promoter to
get specificity for Beta cells expression, then the two coding sequences for Oaf1p and Pip2
separated by a 2A viral peptide; and on the other hand, the promoter sequence of both TF, a
minimal promoter that has been proven to improve its response and the Cre recombinase.
Beta cell specific expression of the proteins and in the presence of fatty acids they will bind to
the promoter and start transcribing Cre.
Diapo 9.- To be easier to handle, we decided to insert all the structure in just one construct,
containing a neomycin resistance cassette, that would insert in one of the Ins1 alleles by
homologous recombination, disrupting it, but since there is haplosufficiency for this gene,
theres no problem. The mouse strain that well be using is already homozygous for floxed
p38alpha.
Diapo 10.- To get the desired mutant mouse, we start by culturing ES cells from the same
strain and then doing an electroporation to deliver the vector with our construct and then
adding the neomycin to the medium to get positive selection. Once we get the resistant cells
we have to genotype and select the one that inserted properly the construct. In a separate
step we get a 2-cell stage zygote and expose it to Electrofusion to get a tetraploid blastocyst.
We do the injection of the obtained ES cells, and then transfer it to a pseudopregnant female.
Finally, with that mouse we have to cross it with a homozygous floxed p38alpha and half of the
progeny will be transgenic, being the mouse that we want to test with.
Es un enfoque que refleja el papel de p38alpha durante el estmulo de cidos grasos altos y no
antes, ya que hasta ese momento, es igual que el WT, relacionando ms directamente el
estmulo y la respuesta (que en WT es apoptosis), y no por otras causas que tambin puedan
activar a p38alpha. Desactivar p38 en el momento el que tendra que actuar y ver qu ocurre.
Para ello, decidimos disear un sistema inducible en el que, al haber altas concentraciones de
cidos grasos y no antes, se expresara la protena Cre y eliminara el gen p38alpha. En
mamferos, la regulacin del metabolismo de cidos grasos presenta protenas que son
capaces de detectar los niveles de dichos cidos grasos y activar la expresin de protenas
relacionadas con el mismo, como son PPAR y RXR. Estas protenas tambin intervienen en la
va de sealizacin que afecta a p38, por lo que no podamos utilizarlas. Para solventar este
problema, tomamos prestado el sistema homlogo a PPAR y RXR que utilizan las levaduras,
Saccharomyces cerevisiae para as evitar crosslinkings. En este caso las protenas a utilizar son
Oaf1p y Pip2.
Para llevar esto a cabo, partimos de 129/SvEvBrd (Lex-1) ES cells de ratones que ya tienen
ambas copias del gen p38 alpha floxeados. En ellas haremos gene targeting en dos pasos.
Mediante electroporacin: Primero la construccin con Ins+Oaf1p+Pip2 (cromosoma 19) con
resistencia a la neomicina, por HR en el gen endgeno donde est presente Ins1 de forma que
quede +/mutIns1; Seleccionamos los que presentan resistencia y repetimos el proceso:
segundo la construccin con ORE+Cre en el locus ColA1 (cromosoma 17) con resistencia a la
puromicina, quedndonos +/OREmut. En ambos casos queda una copia del gen endgeno para
evitar complicaciones.
Una vez hecho esto podemos proceder de dos formas: utilizar un blastocisto tetraploide
mediante electrofusin cuando tenga dos blastmeros, para asegurarnos que el embrin
desarrollado slo proviene de las ES cells modificadas, o generar una quimera y realizar dos
cruces para obtener los ratones que tengan la mutacin en la lnea germinal. En el caso de
hacer los cruces debemos de caracterizar y genotipar la descendencia para seguir teniendo
siempre heterocigotos de ambas construcciones.