Vacas transgnicas

modificadas con miRNA

para producir leche
hipoalergnica
Jess Blanco Piar

4 Grado en Bioqumica
Biologa Molecular aplicada a la
alimentacin.

INTRODUCCIN

Leche de vaca: composicin proteica distinta a la humana

2-3% nios presentan alergias

BLG-A

Transporte de
molculas
hidrofbicas
Hipocolesterolemia
Propiedades
antioxidantes

-lactoglobulina (BLG): principal alrgeno

BLG-B
Funcin especfica
desconocida

Mota HC, Pinheiro JA, Aguilar AA. Alergia a protenas do leite de

vaca. Acta Pediatr Port. 1999;30:1916.

Dias A, Pinheiro JA, Santos A. Persistence of cows milk allergy beyond

two years of age. Allergol Immunopathol (Madr). 2010; 38(1):812.
https://elgustodesaber.wordpress.com/category/innovacion-alimentaria/

INTRODUCCIN

Potencial
alergnico

Enfoques ms
directos

Hidrlisis
enzimtica

Claudia Merkl, Simon Leuchs. RNA

Interference in Pigs. Mol Biotechnol
(2011) 48:3848.

siRNAs
shRNAs
miRNAs

RNAi:
Mayor regulacin

LGB-KO:
Recombinacin
homloga

Control espaciotemporal de la
expresin

Yu et al (2011)
Modificacin
biallica del gen
LGB

Antigenicidad
residual
Sabor amargo
Coste

Shengli Yu, Junjie Luo, Zhiyuan Song. Highly efficient modification of

beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell
Research (2011) 21:16381640.

2012

Produccin de leche hipoalergnica

INTRODUCCIN

BLG-KNOCKDOWN: miRNAs especficos

Vacas transgnicas: coste

Becerro transgnico:
miRNAs validados in vivo

BLG-KD:
Expresin de miRNA y BLG

Clulas en cultivo:
evaluacin miRNAs

BLG-Knockdown en clulas cultivadas

BLG-B: variante conservada

Exclusin de miRNAs con

homologa frente a secuencias
off target
Anower Jabed, Stefan Wagner, Judi McCracken. Targeted microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS 2012 109 (42) 16811-16816.

Problema:

Expresin de BLG
limitada al tejido
mamario en lactacin
Plsmido
BLG

Cotransfeccin:
Clulas COS-7
GFP
miRNA

https://www.mirusbio.com/applications/cotransfection

Plsmido
bicistrnico

BLG-Knockdown en clulas cultivadas

WB: expresin BLG normalizada frente a GFP

Efectos acumulativos: miRNAs en tndem

Anower Jabed, Stefan Wagner, Judi McCracken. Targeted microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS 2012 109 (42) 16811-16816.

Evaluacin in vivo
Anower Jabed, Stefan Wagner, Judi McCracken. Targeted microRNA
expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS 2012 109 (42)
16811-16816.

90-98% actividad
knockdown

BLG-Knockdown en ratones
http://www.nature.com/onc/journal/v24/n29/fig_tab/1208557f1.html

Reemplazamiento promotor constitutivo por promotor WAP

Eliminacin GFP: efecto en la funcin endgena

WAPmiRNA 4-6

miRNA 4-6

-globina
de pollo

BLG
Knockdown persistente?

Anower Jabed, Stefan Wagner, Judi McCracken. Targeted

microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS
2012 109 (42) 16811-16816.

Anower Jabed, Stefan Wagner, Judi McCracken. Targeted

microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS
2012 109 (42) 16811-16816.

4
embriones

BLG-Knockdown en ganado

Fibroblastos fetales bovinos: transferencia nuclear

http://www.nature.com/nbt/journal/v21/n2/full/nbt783.html

Transfeccin con cassette de expresin miRNA 4-6: 5 embarazos

Clona 312/3

Anower Jabed, Stefan Wagner, Judi McCracken. Targeted microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS 2012 109 (42) 16811-16816.

Anower Jabed, Stefan Wagner, Judi McCracken. Targeted microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS 2012 109 (42) 16811-16816.

BLG-Knockdown en ganado

Induccin hormonal de la lactacin

Knockdown EFECTIVO

Anower Jabed, Stefan Wagner, Judi McCracken. Targeted microRNA expression in dairy cattle directs
production of -lactoglobulin-free, high-casein milk. PNAS 2012 109 (42) 16811-16816.

CONCLUSIN
BLG:
Funcin
desconocida

Falta de
modelos
murinos

La
especificidad
de los miRNA
no se
relaciona con
su eficiencia

https://elgustodesaber.wordpress.com/category/innovacion-alimentaria/

Intentos
infructuosos
KO

Estrategia
Knockdown
basada en
RNAi

Alteraciones
fenotpicas?
Becerro sin
cola

https://img0.etsystatic.com/061/1/6771567/il_570xN.775783168_8peh.jpg

La produccin de vacas lecheras que sufren un knockdown

de BLG puede permitir en el futuro examinar si la leche
producida por estas vacas tiene menor potencial alergnico
que la leche normal

BIBLIOGRAFA

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