Ligand Activated Site Specific Recombination Of Gene And The Generation Of Transgenic Mice

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

Rudolph Jaenisch bred the first genetically modified mouse in 1974. Today, millions of mice are used in genetic modification experiments. (Genetic Modified Mice) Rudolph showed that foreign DNA could be integrated into the DNA of early mouse embryos. Despite the success of the transgenic mouse, their results in mice may not breed the same for humans. The mouse is small, making it an economical choice, and it also breeds very well. Therefore, mice were disposable. Scientists can use mice without hesitation because there is

Explain why parabiosis of an ob/ob mouse and a lean control resulted in partial normalization of body weight in the ob/ob mutant mouse.

Flow Diagram Homework Assignment Name: Preetam Ganti Using the table below, explain the purpose of each of the steps of the Experimental Flow Diagram (Figure 1 from your lab manual – it is provided on the next page) for answering the two research questions guiding this laboratory investigation: 1. Is the bacterial contamination at these three farms due to the same plasmid or different plasmids? 2. What is the frequency of tetracycline resistant bacteria in the beef farm cultures?

A sample of three female students at the University of Illinois at Urbana-Champaign was involved in this research where cheek cells were extracted from gargled saline solution and placed in a 15-milliliter tube to centrifuged for two minutes at maximum speed to form a pellet at the bottom. These cells will undergo Polymerase Chain Reaction (PCR) amplification technique to generate billions copies of a particular deoxyribonucleic acid (DNA) sequence so we can increase the success rate and clarity on the gel electrophoresis and sanger sequencing. The tube was then emptied to only have the pellet remaining and 500 microliters of Chelex stock was added to the tube to bind metal ions that can interfere with PCR reaction. The tube was then closed loosely, placed in boiling water for ten minutes to lyse the cells, shaken, then centrifuged for a second time for two minutes. Three microliters of clear supernatant from the Chelex solution was added to the primer to help bind polymerase to the gene of interest during annealing in PCR amplification. Primers were used in the solution for our restriction enzyme, Haelll, to cleave our DNA sequence at the GGCC location, between the guanine (G) and cytosine (C).

The creation of a transgenic animal in this case a mouse can be divided in four crucial steps. First the development of the transgenic construct, second its preparation for integration with the pronucleus of fertilized mouse embryo, third screening and identification of transgenic founder mice and last successful breeding of founder

1.Create targeting construct using recombinant DNA technology. The construct must contain the transgene DNA sequence and the promoter necessary for gene expression. The taransgene has to be flanked by long segments of DNA homologous to endogenous mouse genome. The targeting construct must also contain positive selection gene between the homology arms and a negative selection gene outside of the homology arms.

Blood samples were obtained from all family members. Genomic DNA was extracted from 100-μl samples of peripheral blood mononuclear cells incubated with proteinase K in lysis buffer, followed by phenol/chloroform extraction and ethanol precipitation. Finally, the pellet was dissolved in 30 μl sterile water and stored at -20°C until further tested. To determine mutation, specific primer sets within the APC gene were designed (primer sequences upon request). Two microliters of DNA were used to set up 25-μL PCR reactions by using the Perfect Taq plus MasterMix (5 prime GmbH, Hamburg, Germany). The amplification reaction was carried out under the following conditions; pre-incubation at 95°C for 3 min, followed by 35 cycles comprising denaturation at 95°C for 30s, primer annealing at 55°C for 30s, and extension at 72°C for 1min, and concluded by a final extension at 72°C for 5 min. Upon electrophoresis in 2.0% agarose gel stained with EtBr, the amplification products were visualized under UV

2. McClean, Phillip. (1997). Cloning and molecular analysis of genes: Polymerase chain reaction (or PCR). Retrieved on December 6, 2014, from http://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/ clone9.htm. 3.

Results: Enzyme digestion analysis, PCR and DNA sequencing results showed the target gene was inserted correctly into the vector. The expressed protein was purified successfully via Ni-NTA affinity chromatography and its molecular weight

Gene Knockout is a genetically engineered technique in which an organism is modified to carry genes that are non-functional. The non-functional gene results from the insertion of a foreign sequence that disrupts or stops the genes functionality. The gene will no longer code for the specific metabolic processes and may cause serve implications on the organism. The method of gene knock begins in a lab with a test tube of plasmid, DNA construct proceeding to cell culture. The end goal is to create transgenic animals that carry the altered gene. Gene Knockout can be passed down through future generation if the embryonic stem cells are modified and inserted into an embryo in the early stages of development.

A reverse transcription PCR (RT-PCR) procedure will be carried out using the designed primers for cDNA. The cDNA is obtained from the mRNA and not the genomic DNA. Derived cDNA will not be manipulated to obtain sticky ends, rather it will be blunt-ended.

The possibility of human genome editing was not exactly promising until a few limitations were set aside. Patrick D Hsu, Feng Zhang, and Eric S Lander are all very qualified specialists of biology at the broad institute of MIT and Harvard. In their report, they discuss the limits with genome editing in eukaryotes specifically: “Eukaryotic genomes contain billions of DNA bases and are difficult to manipulate. One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination (HR), which integrates exogenous repair templates that contain sequence homology to the donor site” (Hsu) Here, the scientists recognize some trouble with altering DNA bases. Though after further study, the biologists soon recognized that homologous recombination was quick fix to this issue. With a big limitation out of the way, potential application of this new technology was a new focus. The scientists concluded with the ability for CRISPR technology to underlay the key factors in order to produce new drug targets, as well as directly altering harmful mutations through genome editing. The ability to understand the

Background: Polymerase Chain reaction (PCR) is an in vitro process that enables exponential amplification of the define target DNA sequence. The process involves three steps: denaturation, annealing and elongation. In the first step, the reaction mixture containing target

Taq Polymerase is essential in Polymerase Chain Reaction(PCR) experiments to obtain a PCR amplification of an unknown gene. The unknown gene is then ligated into a vector plasmid, which is placed in a bacterium Escherichia Coli to transform and multiply. Ultimately, identification and characterization of the unknown gene is done using electrophoreses and gel imaging. Cloning techniques such as the one performed have been used for many years to isolate genes from a variety of species. Isolation of unknown genes serves many purposes. Nucleic acid sequence of the unknown gene can be derived which can be compared to other known gene sequences and the function of the gene can be derived. The clone made can be used to study the