BMP2 Knockout in Xenopus laevis embryos Nathaniel Smith Birth Defects Cause 20 of infant death Birth Defects Birth Defects Child Health USA 2014 2014 Birth Defects Remain ID: 912896
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Slide1
Understanding Cleft Lip and Palate via BMP-2 Knockout in Xenopus laevis embryos
Nathaniel Smith
Slide2Birth DefectsCause 20% of infant death“Birth Defects.” Birth Defects, Child Health USA 2014
, 2014.
Slide3Birth Defects
Remain
a mystery
?
?
?
Slide4Cleft Lip and PalateOne of the Most Common Birth Defects
Bloomfield, V., & Liao, C.H. (2015)
Cleft lip
Cleft Palate
Slide5Cleft Lip and PalateNO CURE!!!
Bloomfield, V., & Liao, C.H. (2015)
Slide6Cleft Lip and Palate Huge Implications
Jugessur
, A., & Murray, J. C. (2005)
Slide7Cleft Lip and PalateCan be understood Using Xenopus laevis
Slide8BMP-2 GeneMost altered Gene in Xenopus
with cleft palate
Wahl et al (2018)
Slide9BMP-2 GeneMicrodeletions of BMP2 cause cleft palate in humans
Sahoo
et al (2011)
Slide10BMP-2 GeneKnown for bone and cartilage formation
Sahoo
et al (2011)
Slide11Experiment - Knockout of BMP-2 GeneHelp Determine causes of Cleft Palate
Slide12Knockout of BMP-2 Gene
Using CRISPR/Cas9
Slide13Knockout of BMP-2 GeneDesigning the sgRNA
Slide14Knockout of BMP-2 GenePicking a high-quality sgRNA
Slide15Knockout of BMP-2 GenePerformed at 1-cell stage
Slide16Knockout of BMP-2 GeneMicroinjection of CRISPR
Slide17Knockout of BMP-2 GeneEmbryos incubated for 3 days, until facial development occurs
Slide18Assess Mutations in BMP-2Done via DNA Extraction and PCR analysis
Slide19Measuring gene expressionWnt1 and RARg are the genes measured
Wahl et al (2018)
Slide20Measuring gene expressionDissection of Frogs Faces
Slide21Measuring gene expressionIsolating RNA from tissue sample
Slide22Measuring gene expressionRT-PCR and qPCR can help us do this
Slide23RT-PCRRegular PCR, but with RNA and new enzyme
Slide24RT-PCRDesigning high-quality primers
Slide25RT-PCRDesigning high-quality primers
Slide26RT-PCRcDNA synthesis
Slide27RT-PCRcDNA synthesis
Slide28qPCRSame as PCR, but fluorescent based dye is used
Slide29qPCRFluorescence is measured between each thermal cycle
Slide30qPCRCt value represents flourescence
Slide31qPCRHigh Ct value = Low Gene ExpressionLow Ct value = High Gene Expression
Slide32Delta-Delta Ct AnalysisUses Ct values to quantify gene expression
Slide33Delta-Delta Ct AnalysisUses Ct values to quantify gene expression
Slide34Potential results of BMP-2 KnockoutIncreased/Decreased Wnt1 & RARg
Slide35Potential results of BMP-2 KnockoutBlue – Increased, Red - Decreased
Slide36Potential results of BMP-2 KnockoutUncovers interaction of genes responsible for Cleft Lip and Palate
Slide37Potential results of BMP-2 KnockoutUncovers interaction of genes responsible for Cleft Lip and Palate