A Research Study On Protein Guided Injections, And Transcription Activator Like Effector Nucleases ( Zfns )

Title: Cloning of Enhancer of Zeste Homolog 2 in forward orientation into Escherichia Coli using histidine-tagged pbluescript II KS+.

Speciation is when a population cannot exchange genes with another population. This occurs because of geographic, behavioral, and temporal isolation. The rodents in the St. Kitts island is a small species that has been threatened by development. The populace is extremely minuscule and the rodents are struggling in finding a mate. During my study, I noticed the Nevis rodent population were very healthy and could be used as potential mates for the rodents in the island of St. Kitts. Unfortunately, after 240 attempts to mate the populations no reproductive event was successful. The St. Kitts rodents and the Nevis rodents cannot exchange genes

Majority of mice in New Mexico’s Valley of Fire have dark color. The population increase of dark-colored mice in Valley of Fire happened because of natural selection. Mice that do not suit the environment, which are light-colored mice, cannot survive in Valley of Fire because they are easily noticed on dark soil by the predators. On the other hand, dark-colored mice can hide easily on dark soil, which made them survive. Whereas the mice near the desert are mutated, those near the lava are not mutated. Each mouse from different lava found to have a different gene. Whereas the mice near the desert’s color change was from a random mutation, that of the mice near the lavas was not random. Because, each dark-colored mouse from two different lavas

This lab had 2 exercises. Exercise 9.1 involved observing pictures of 60 F2 offspring and recording the phenotypes for 6 different traits. Exercise 9.2 required us to perform the “chi-square test” to determine whether the data we collected matches the standard Mendelian ratio.

The Rock Pocket Mouses’ coat color is extremely important because the mice use it as a mean of camouflage if the mice are a different color of fur than its environment, their overall fitness, how well an organism can survive and reproduce in its environment, decreases. Due to a volcanic eruption, most of the environment has turned from a sandy-colored soil to black soil; therefore making it very difficult for the unmutated, pale mice to survive. The dark fur color of the Rock Pocket’s population occurred due to a random mutation, a mistake made in the DNA whether it is good or bad, known as the MC1R gene. Because of this, variation among the mice was created, making some mice dark-colored and others to stay light-colored. Dark-colored mice

After we did our research on the mice, there was a drought, causing lots of environmental changes. One of these changes was the grass perished leaving large dirt patches. This let the brown mice camouflage. In contrast, the gray mice were found very easily by predators and later consumed. Because of this, the ratios of gray mice decreased (As shown on the right). Naturalist Brett Holland also got similar results (As shown below).

The next two questions, “Why study bacterial gene regulation?” and “What are the basics of gene expression?” were addressed next in the seminar. Fascinatingly, a bacterial cell knows how to respond based on their environment. For example, they recognize and respond differently when in water than in a human. The average bacterial cell has close to 1000 genes, this is significantly more genetic information that they can use at any given time. These genes are stored in the DNA and each individual gene provides the information to make an individual product. So, how is a gene expressed? The first step in this process is called transcription. During transcription, the information encoded in the DNA is copied by RNA Polymerase to make mRNA. The next step is referred to as translation. During translation, the information in the mRNA is “translated” to make a protein using the ribosome. These steps basically turn genes “on and off” in a bacterial cell in response to a change in their environment. How does this happen? According to Dr. Henkin in her 2008

They discovered full dominance in the homozygous DD allele, and an intermediate dominance in reflection in the heterozygote. Hoekstra et al (2014) found that the allele frequency of the Mc1r allele varied in accordance to the surrounding substrate the mice inhabited. There was strong selection against light mice found on a dark substrate as opposed to darker mice found on a lighter habitat. When looking at migration, Hoekstra et al (2014) had a few concerns. First, they feared the migration was unrealistic, despite the lava region functioning as islands. They also had concerns that new alleles were introduced through immigration among untested sites and the tested locations. Hoekstra et al (2014) concluded that immigration occurred more often than emigration among the populations by using their estimated migration patterns. Because of the selection against light mice on dark substrate, Hoekstra et al (2014) concluded that the light colored mice had adapted the melanic mutation as a cryptically colored, antipredator

A simple but effective method is correctly designing gRNAs. Ideally, the target sequence must be a unique and nearly perfect homology to the gRNA with as little to no homology at other points within the gene sequence. Another potential strategy involves reducing the concentration of gRNA and Cas9 that are expressed in cells. However, this strategy is considered inconclusive due to inconsistent reporting. Some cell lines showed a large decrease in rates of off-target binding while others showed no effect. An increasingly promising method for improving specificity involves using a mutated nickase version of the Cas9 enzyme. This modified version contains only one catatylic domain and is used as a single nickase system capable of cutting a single strand of DNA. This results in a “nick” or single stand break rather then a DSB and is repaired by HDR. To further reduce the risk of off target binding, a double or dual nickase CRISPR system can be implemented. This system uses two nickases which bind to opposite strands of the target gene, generating a DSB. This system closely resembles the Fokl enzyme activation used in TALEN, in that off target effects are reduced because the probability of two off target nicks within the same region is very unlikely. When the DSB is made, activation of NHEJ is initiated; to further increase specificity and reduce off-target effects, the HDR mediated editing is advised for both nickase

When reading the experimental design, the grouping was not clear. How many mice? How many groups? I expected to see that

The purpose of this experiment was to study the effect of an experimental drug on the number of offspring a mother mouse has. The question asked was: “Does the experimental drug affect the number of the offspring a mouse has?” The hypothesis was: “If the experimental drug is given to mother mice, then the number of offspring in their litters will change because of the chemicals in the drugs.”

Each litter has up to six babies, helping the population increase dramatically in a short period of time. At least there are two genes involved

Before the transgenic rat embryos are generated, pseudo-pregnant females must be prepared. This is done by setting up fifteen mating cages where there is a wild-type female rat with a vasectomized male rat. The rats must have mated a day before the insertion of embryos because this will allow the female rat to be in the prime hormonal condition to receive the embryos for a pregnancy. After a day has passed, the female rats are checked for the presence of a mating plug, which indicates that the rats have mated6. The mated females then undergo a surgery that implants the transgenic embryos with the female rats. Once pregnancy has passed and the rat pups have been delivered, the rat pups must be monitored to make sure that they are growing normally to rule out that inserting the HaloX gene could be affecting their growth. Once it has determined that the rats are growing healthily, it is important to see if the inserted HaloX gene has been implanted successfully before beginning the pain

Humans and mice share similar pathological and physiological features in immune, cadiovascular, nervous, endocrine and other systems1. Manny similarities are also found when comparing their genomes. In addition to those similar features, mice can be easily manipulated genetically2, therefore, making them attractive in becoming models for biomedical research. Manipulation of mice genetically allows generation of human pathologies models. These models can then be used in biomedical experiments to understand the underlying mechanism of certain human diseases. Mice are also small in size, highly prolific (with eight to twelve pups in each pregnancy), and have a short generation time of eight to nine weeks. The biggest advantage is that their genomes are highly conserved3. As such, many mouse mutants were generated in the early days by exposing the mice to DNA-damaging agents or radiation4. However, these approaches create many

RiboTALE networks studied were comprised of two modules and each module was based on a separate plasmid. One of the modules, repressor module, expresses fusion of riboswitch (RS) from an inducible PBAD promoter, second module, target module, contains a TALE binding site (TBS) downstream of a regulated or constitutively expressed promoter. Ribosome binding site (RBS) and gfp are present downstream to TBS (Fig. 1). gfp was used as proxy to quantitate the promoter expression. Repressor modules were based on kanamycin resistance medium copy number BioBrick plasmid, pSB3K3 (Shetty et al, 2008). Four different types of repressor modules, PR1T1, PR1T8, PR2T1, and PR2T8, were constructed combinatorially by implementing two types of riboswitches and two types of TALEs (supplementary table 1). Used riboswitches, Riboswitch-1 and 2, were theophylline inducible (Lynch & Gallivan, 2009; Topp et al, 2010). TALES with either low dissociation constant (Kd = 1.3±0.3, T8) or with high dissociation constant (Kd= 240±40, T1) were selected (Meckler et al, 2013). Each of the TALEs recognizes a specific 19 bp target DNA sequence.Two families of target modules were constructed. One of the modules was based on regulated tetracycline promoter, PTet, and other was based on a family of constitutive promoter, PConst, generating different level of outputs. Target modules contain two types of TALE binding site, TBS1 or TBS8. TALE1 binds with TBS1 and TALE8 binds with TBS8 (Fig.1). All measurements were