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BICD 101 Lab Report #2

BICD 101 Lab Report #2



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Published by mwjackso
This lab report involved the use of mouse ES cells and was done during my senior year at UC San Diego.
This lab report involved the use of mouse ES cells and was done during my senior year at UC San Diego.

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Published by: mwjackso on Apr 04, 2009
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Lab #2: Reverse GeneticsA02-92-6779- 1 -
Lab Report #2
Reverse Genetics:
 Creation of a Mouse Model for a Human Disease via theTransfection and Growth of Mouse Embryonic Stem CellsMichael Wade JacksonA02-92-6779BICD 101: Eukaryotic GeneticsMay 29
2003Group 6:Michael JacksonSusan Liem
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
Lab #2: Reverse GeneticsA02-92-6779- 3 -in the 3.5 day old embryo of a mouse (blastocysts). The stem cell is harvested from thedeveloping mouse embryos a short time after fertilization and before significant zygotedevelopment into a viable embryo. The ES cells are maintained in culture via the use of embryonic fibroblast cells (EF cells) which have been immortalized (halted in mitoticdevelopment) to provide nutrients and growth factors to keep the ES cells fromdifferentiating. The first introduction of a germ line mutation in ES cells was in 1984.The vector for introduction of the mutation was a retroviral vector. This vector was thenused to incorporate the mutation into a blastocyst and the production of an agouti mousewas the result. This mouse was a chimeric mouse that when the progeny of the F1 wereselfed the F2 generation produces
homozygous mutants which have two copies of themutant gene creating an organism that can become a model for human disease.In 1987 the first gene targeting and knock out experiment was carried out inwhich the procedure produced a method by which a normal gene could be replace with agene of interest using the DNA repair machinery of the host cell. The creation of thisknockout technique now allows for the ability to create mouse models of human diseasewith a higher degree of efficiency. The knockout technique once perfected led to thecreation of skid mice (severe immune compromised) which are models for the humanimmunodeficiency virus (HIV). The technique used in these labs to create a mousemodel for a human disease is transfection by electroporation of a linear targetingconstruct.The goal of these labs is to produce mouse models of human disease. This process is made possible by advances in cellular manipulation and culture since 1980 thathave paved the way for the techniques of today. The major issue surrounding the procedures and techniques used in this educational lab are the irregular sequence of events that are not in sync with the actual progression in a diagnostic lab environment.However, the techniques learned in this lab are the current and frequently used techniquesthat will give the students of this class the ability to compete in a genetic biology lab and provide the background to understand the process of gene targeting.

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