This is the script from my presentation about this essay and also my plan for it. Hyphotesis The Mega Bcl protein is overexpressed in a transgenic cell, it would prevent apoptosis and also increase the likelihood of developing cancer. The Drug BH3 mimetics inhibits the anti-apoptotic protein Mega-Bcl from proliferating throughout the mitochondria as well as increase levels of the BH3 pro-apoptotic protein. Aims : Create the transgenic mouse model that is overexpressed with the Mega-Bcl protein knocked in. To determine if the transgenic mouse models expression promotes a cancer phenotype and how its changing expression of mice’s health To test the efficacy of BH3 mimetics drug in vitro and vivo use the tumour model Transgenic Mouse Model : We use Knock in Model Artificially altering the expression of an already present or induced gene Investigate the function of the gene, what does it do ? how does the phenotype change after expressing or losing expression the gene Is it going to be loss of function or gain of function ? Methods of producing transgenic mouse models Transforming embryonic stem cells (ES cells) growing in tissue culture with desired DNA; Make the DNA Using recombinant DNA methods, build molecules of DNA containing The gene you desire which is overexpressed it with Mega bcl- protein Vector DNA to enable the molecules to be inserted into host DNA molecules Promoter and enhancer sequence to enable the gene to be expressed by host cells. Transfrom cells in culture Expose the cultured cells to the DNA so that some will incorporate it. > Select for successfully transformed cells > inject these cells into the inner cells mass (ICM) of mouse blastocysts. Embryo transfer Prepare a pseudopregnant mouse (by mating a female house with vasectomised male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive. Transfer the embryos into her uterus. Hope that they implant successfully and develop into healthy pups ( no more than one-third will). > Test her offspring Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10-20% will have it, and they will be heterozygous for the gene. > Establish a transgenic strain Mate two heterozygous mice and screen their offspring for the 1 in 4 that will be homozygous for the transgene. Mating these will found the transgenic strain We use this method for 40 mice 20 mice WT 20 mice are transgenic cell that have protein in it (Mega Bcl-Protein ) Phenotypic effect measurement Weight of the animal ( represent a clear indication of health and imaging ) Survival Graph ( represent the mortality rate) As a result shown from these techniques the transgenic expression its tumour oncogene Drug efficacy in Vitro We use Caspase Activity to test the drug efficacy We will test the drug based on 2 cell HL-60 & HL-60mega bcl2 Treated with Drug name is BH3 mimetics Non treated Drug efficacy in vivo tumour model Testing your drug in a mouse model Remember Control Since the drug efficacy was proven by the caspase assay in vitro, we administered our BH3 mimetic drug in to vivo tumour model to test the efficacy of the drug. Tumour growth was measured in the mouse models an was evident in those that were overexpressed with Mega Bcl protein and untreated with BH3 mimetic whereas tumour size reduce in mouse models that were overexpressed with Mega bcl when treated with the BH3 mimetic protein Subcutaneous Model ( Inject cancer/ transgenic cells under the skin to form tumour ) Administer Protein ( Measure tumour growth and body weight ) Tumour size ( caliper measurement ) 6 mice all genetically similar injected with tumour cell subcutaneously 1 pair = control, 2 other pairs are experimental groups · Control = without BH3 mimetics drugs , test group 1 = with a little amount of BH3 mimetics drugs , test group 2 = High amount of BH3 mimetics drugs · Use Bioluminesence ( using light to image cancer cells( cancer cells express a light producing molecule )
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