Lab #2: Reverse GeneticsA02-92-6779- 2 -
The goal of this series of experimental procedures was to create embryonic stemcells which contained an inserted transgene that would allow for the creation of a mousemodel of a human disease. This model would then be used to analyze and evaluate newtherapeutic approaches to treatment and pathogenesis of the human disease. The primaryeducational goal of this series of experiments was to gain knowledge and practice in cellculture technique which involved: 1) creation, immortalization, and storage of EF celllines 2) Transfection of ES cell lines with linear targeting construct 3) ES cell selectionvia antibiotic resistance 4) Growth of monoclonal ES cell line 5) Analysis of targetconstruct insertion via PCR and 6) Injection of ES cells which exhibit homologousrecombination into blastocyst for the creation of a mouse model after F2 generation in
of progeny. The outcome of these procedures was the production of non-homologousend joining (NHEJ) cells which were amplified in the process of PCR exhibiting thecontrol band at 2000 Kb. The PCR amplification displayed a lack of a candidatemonoclonal cell line that exhibited homologous recombination.
The field of genetics is one that is in a process of continual expansion as the progression of biotechnology and laboratory innovation pushes the boundaries of once asemi-static field into a multitude of arenas blurring the lines between different specialtiesin biology. The methods utilized in these experiments are relatively routine today butwere novel in the 1980’s. The scientific approach that is exemplified in this five week period is the process of reverse genetics. Reverse genetics is the process by which a genein mutated and the phenotypic consequence is then determined. This is a direct oppositeto the process of forward genetics in which a phenotypic mutation is observed and thehunt is on to trace this mutation back to a target gene. The environment in which reversegenetics was used in this class is in the field of mouse modeling of human diseasethrough the creation of homologous recombinant mouse cells to produce chimeric(agouti) mice and then through selfing of progeny produce
homozygous mutants in theF2 generation. The production of ES cells that contained HRE is based of the past work of many scientists and utilized many common techniques practices in labs around theworld.Embryonic Stem Cell (ES cell) technology finds its roots in the early 1980’s whenthe determination was made that ES cells are pluripotent, meaning that these ES cells can become any cell line in the laboratory animal. The 1
isolation of ES cells came in 1981