Having an abnormal number of chromosomes is known as aneuploidy. Aneuploidy opens the window for many possible diseases, and among these are down syndrome, acute lymphocytic leukemia, and acute myeloid leukemia. Normally, baby fetuses have 46 chromosomes; 23 from the sperm cell and 23 from the ovum. However, if there is an abnormal number of chromosomes, there will be complications during the growth of the fetus. Aneuploidy diseases can be deadly but new developing technology would help prevent this and allow for future healthier generations. Currently, there is an experiment that has been done to reverse the damage done by aneuploidic diseases. The study was done with Xo mice, which are mice born with only one X- chromosome. In this study, the aim is to be able to remove an extra chromosome in human cells that have been diagnosed with a trisomy 21 chromosome, a diseases commonly known as down syndrome. If successful, this could be a breakthrough in the genetic engineering field of study and could possibly be a gateway to cure cancer. In this study, the goal is to completely eliminate an entire chromosome in human cells from trisomic cells.
A study was done using mice, specifically with Xo or monosomy X mice which means that they were born with one sex chromosome (Probst et al, 2008). The Xo mice were born with what we call Turner syndrome, which is a disease where a fetus is born without an X-chromosome. One of the differences between the syndrome in mice and humans is that when a woman is born with Turner syndrome, she becomes infertile, unlike the Xo mice, who do not (Probst et al, 2008). They managed to delete this extra chromosome from the offspring. They began by obtaining male mice that had the Y chromosome, which contained inverted LoxP sites, and mated them with female mice carrying the Cre gene. A LoxP site is a site on the P1 bacteriophage consisting of 34 base pairs. The Cre gene creates recombination. Cre recombinase is an enzyme designed to cut out genetic sequences between two LoxP sites (Hu et al, 2015). The loss of the chromosome was ultimately achieved by recombination due to the Cre enzyme presence between LoxP sites on identical DNA strands during the embryonic creation. Apart from the cre inverted LoxP sites, what was also included was a cassette that contained green fluorescent protein (GFP) and drug resistant genes bracketed by a pair of inverted LoxP sites(Sato et al, 2017).
The GFP was added to be used as a visual tag and ensure visibility. A similar process was done to humans. A cassette was made which contained two inverted LoxP sites and human herpes simplex virus thymidine kinase type 1 gene,(HSV-TK). The HSV-TK is a gene that is both added and used as a marker to select specific cells lacking the targeted chromosome (Iyer et al). The way this cassette was added was through the process of homologous recombination, which is a specific recombination where nucleotide sequences are exchanged between two similar or identical molecules of DNA. The technology used is called clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9 nickase system. CRISPR/Cas9 is a family of DNA sequences in bacteria that contain bits of DNA viruses that have previously attacked the bacteria and this process is unique because it allows for alteration of sections of DNA (Questions and, 2017). The process began by using HeLa cells, which are cells that were preserved and were originally taken from Henrietta Lacks, a woman who died of cervical cancer(Dimanno & Spigner, 2017).
These cells were cultured in Dulbecco’s Modified Eagle Medium and gave off fluorescence in situ hybridization (FISH) analyzation, after hybridization was performed on them(Sato et al, 2017). Hybridization is the process of forming a double stranded nucleic acid from joining two complementary strands of DNA/RNA(Savic, 2017). This occurs in a situ, specific site. The next step is plasmid construction, which is a typically circular DNA strand in the cytoplasm that can independently replicate without chromosomes. CRISPR/Cas9 nickase system was used to integrate the DNA cassette into the HeLa genomes. The cre recombinase was used as a vehicle to direct and artificially carry foreign genetic material into another cell. The fifth step is to isolate the genomic DNA from the HeLa cell and to use it as a molecular mold for the polymerase chain reaction (PCR).
The final step is to count the cell viability. It was then concluded that at least 200 cells were counted in each culture after four days. In this experiment, chromosome 21 was targeted in the HeLa cells due to how three dots were fluorescent in situ hybridization (FISH) analysis. HeLa cells have an unbalanced set of chromosomes, which in this case, carried three copies of chromosome 21. The donor plasmid and the constructed DNA cassette were used and integrated first. The donor plasmid introduced nucleic acids into cells with the CRISPR/Cas9 nickase system. Homologous recombination was induced by using the CRISPR/Cas9 nickase system. Essentially, the DNA cassette was integrated between the genes in chromosome 21. As this process continued, Cre recombinase induced the elimination of a chromosome, which was shown in the HeLa cells. FISH analysis helped signal that there were now only two chromosomes left due to the signals in the nuclei. It was also noted that the DNA cassette spontaneously disappeared in the HeLa cells and cre transfected colonies contained cells that had chromosome 21 trisomy successfully eliminated.
In conclusion, the system used allows for the elimination of the chromosome 21, however, only in human cells that have not had spontaneous chromosome loss. Nevertheless, the system was successful and could be useful to help determine the set of causes for chromosome abnormalities using cultured cells. It has created a window to continue genetic engineering research and eventually find a solution to bring about a healthier society.
A professional writer will make a clear, mistake-free paper for you!Get help with your assigment
Please check your inbox
I'm Chatbot Amy :)
I can help you save hours on your homework. Let's start by finding a writer.Find Writer