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From 2010.igem.org

Contents 1 Hassan 1.1 MITF 1.1.1 PAX3 1.1.2 FOXD3 1.1.3 TYRP1 2 Nina 2.1 Site direct mutagenesis on Tyrosinase

Hassan

MITF

PAX3

continued from yesterday:

"The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation.... ....PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf."[http://www.ncbi.nlm.nih.gov/pubmed/19026785]{Also: Figure 7. Schematic Diagram of the UV-Induced Melanogenic Response}

FOXD3

"...The transcriptional repressor FOXD3 is expressed exclusively in the neural/glial precursors and MITF is expressed only in melanoblasts. Moreover, FOXD3 represses melanogenesis....MITF expression begins very early during melanoblast migration and that loss of MITF in melanoblasts causes them to transdifferentiate to a glial phenotype. Ectopic expression of FOXD3 represses MITF in cultured neural crest cells....FOXD3 does not bind directly to the MITF promoter, but instead interacts with the transcriptional activator PAX3 to prevent the binding of PAX3 to the MITF promoter. Overexpression of PAX3 is sufficient to rescue MITF expression from FOXD3-mediated repression. We conclude that FOXD3 controls the lineage choice between neural/glial and pigment cells by repressing MITF during the early phase of neural crest migration." [http://www.ncbi.nlm.nih.gov/pubmed/19403660]

TYRP1

[http://www.ncbi.nlm.nih.gov/pubmed/12136092]

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Nina

Site direct mutagenesis on Tyrosinase

The method for this experiment was according to the procedure described in protocols.

Concentration of tyrosinase in its original vector: 320 ng/ul, wanted concentration is 10 ng/ul.

320 / X = 10 ng/ul

X = 32 this means that I need to do a 1:32 dilution.

Dilution:

1 ul sample of tyrosinase mix with 31 ul H2O.

PrimerF:

125 ng is wanted of the primer.

Added 267 ul H2O to the dry primerF sample according to a manual I recived from the company of the primers. This equals 100 uM.

100 uM = 100*10^-12 mol/ul

MW of primerF: 8903 g/mol

100*10^-12 mol/ul * 8903 g/mol = 8.903*10^-7 = 890.3 ng/ul

890.3 ng/ul / X = 125 ng/ul

X = 7.12 this means that I need to do a 1:7 dilution.

Dilution:

1 ul sample of primerF mix with 6 ul H2O.

PrimerR:

125 ng is wanted of the primer.

Added 259 ul H2O to the dry primerR sample according to a manual I recived from the company of the primers. This equals 100 uM.

100 uM = 100*10^-12 mol/ul

MW of primerF: 8894 g/mol

100*10^-12 mol/ul * 8894 g/mol = 8.894*10^-7 = 889.4 ng/ul

889.4 ng/ul / X = 125 ng/ul

X = 7.1 this means that I need to do a 1:7 dilution.

Dilution:

1 ul sample of primerR mix with 6 ul H2O.