Genetically modifying animals is a tool that can be used to increase livestock production. The gene editing technology CRISPR has expanded the possibilities of gene editing. The Cas9 nuclease creates a double strand break which can be repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR). The goal of this experiment was to create a single nucleotide change using the CRISPR/Cas9 system in combination with a single strand oligo nucleotide (ssODN). The single nucleotide that was targeted occurred naturally in Holstein cattle, and is associated with increase milk production. There are multiple factors involved in the Holstein cow's ability to produce large amounts of milk. A mutation in the alpha lactalbumin sequence (α-LA) is one of these factors. The α-LA gene sequence in some Holstein cattle contain an adenine the at (+15) position which corresponds to transcriptional start point of α-LA (+1), while other breeds have a guanine at that position. The adenine in the +15 position of the α-LA gene has been associated with increased milk production. MAC-T cells and Angus fetal fibroblasts were transfected with a Cas9 plasmid, pSpCas9(BB)-2A-GFP, and a ssODN to insert the desired mutation. Using the CRISPR/Cas9 system we were able to create a double strand break resulting in indels and deletions at the (+15) site in MAC-T cells, but we were not successful in creating a single nucleotide change. However, we did see a single nucleotide change in Angus fetal fibroblasts using CRISPRs and ssODN. Following the success of inserting the mutation into a cell line we attempted to create an embryo containing the single nucleotide change using sperm-mediated gene transfer (SMGT). Naked DNA binds naturally to sperm, and can be used to produce transgenic offspring in pigs and cattle. In this experiment, we analyzed methods to select thawed bovine sperm, and evaluated the binding of exogenous DNA to those sperm. Liposome preparation was done using a cationic lipid, 3-(trimethyl ammonium iodide) 1,2 dimystryl-propanediate (TAID) and a neutral lipid, L- Dioleoyl phosphatidyl-ethanolamine (DOPE) prepared according to given protocol. Percoll or swim-up methods were used to select sperm after thawing, followed by incubation (1h or 3h) with the liposome-DNA complexes. We used enhanced green fluorescent protein (eGFP) in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analyzed: 1) no incubation, no liposomes and no DNA, 2) incubation with no liposomes and no DNA, 3) incubation with liposomes and no DNA, 4) incubation with liposomes and 1 ng of DNA and 5) incubation with liposomes and 10 ng of DNA.Once the liposomes had been complexed with DNA they were incubated with sperm for one or three hours before IVF. The CASA results for total motility and rapid motility were significantly different from the control (P<0.01) between the control and the other treatments in the Percoll group as opposed to swim-up. These results confirm that the sperm selected with swim-up has less effect on sperm motility than Percoll. Real time PCR was able to detect plasmid GFP DNA in the DNA of sperm samples and pictures taken of the sperm using the Spatial Light Interference Microscopy (SLIM) confirmed the presence of liposomes on the sperm head and tail. SMGT was then used with IVF to deliver two plasmids containing GFP encoding region under the control of CMV promoter; pIRES2-EGFP and pSpCas9(BB)-2A-GFP for gene transfer. The blastocyst rate was low and ranged from 0 to 20%. Although we did show that plasmid DNA was present on the sperm, we were not able to detect any positive GFP 8 day embryos using SMGT.
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