An Sci 361 1st EditionExam # 1 Study Guide Lectures: 1 - 9All PowerPoint presentations, discussions, reading assignments, and quizzes are requiredLecture 1 (January 23)Animal Biotechnology (lecture and movie)What is somatic cell nuclear transfer? Somatic Cell Nuclear transfer (SCNT): Transferring the nucleus froma somatic cell to an unfertilized oocyte that has been “enucleated” by removal of its own haploid set of chromosomes. In order to begin the development process, the donor nucleus must be fused with the egg through the administration of a brief electrical pulse, and then the egg is activated though exposure to short electrical pulses or a chemical fusion process, after which the embryo starts to divide as if it had been fertilized. These “reconstructed” embryos are typically cultured in petri dishes for 5-7 days until they reach the blastocyst stage. In the case of mammals, the embryo is then placed into the oviducts or uterus of a surrogate or “recipient” dam where it will develop until birth.Embryo splitting: When spontaneous, is the way in which identical twins are formed. When artificially induced, it is cloning. Bisecting a multi-cellular embryo at an early stage of development to generate clones or “twins.” Occurs naturally or can be performed in a lab. Limitation on number of clones that canbe produced, usually two, from each embryo and the genetic merit of the embryo is unknown.Understand why Dolly was different than previous cloned animals? She was the first animal to be cloned via SCNT from a differentiated somatic cell derived from an adult. This result opened up the possibility that clones could be produced from a potentially unlimited number of cells from an adult animal. In the case of clones it appears that the reprogramming of somatic cell modifications is sometimes incomplete leading to inappropriate patterns of DNA methylation, chromatin modification, and X- chromosome inactivation in the developing clone. This can result in aberrant gene expression patterns and correspondingly high rates of pregnancy loss, congenital abnormalities, and postnatal mortality.Understand the difference between genetic engineering and cloning: Genetic engineering (GE) is the manipulation of genetic material (ie, DNA or genes) in a cell or an organism in order to produce desired characteristics and to eliminate unwanted ones. A clone is not completely genetically identical, as there are small differences in the genetic make-up just as there are with identical twins.Be able to give examples of the use of transgenic animals: Cloning offers the opportunity to make GE or transgenic animals more efficiently from cultured somatic cells that have undergone precise, characterized modifications of the genome. Genetically engineered animals are sometimes referred to asgenetically modified organism (GMO), living modified organism, transgenic, or bioengineered animals. The first method to produce GE animals was microinjection of rDNA into blastocysts to produce transgenic mice in 1974.Main objectives of Ag applications for transgenic livestock technology: Improved food products, improved animal welfare, reduced environment footprint.Lecture 2 (January 26)Bovine Leukocyte Adhesion Deficiency (BLAD) and CLADUnderstand how amino acid changes can lead to the disorder in cattle (BLAD): Most calves die within 1 year of age. Deficiency in leukocyte surface glycoproteins known as: integrin’s. Integrin’s: family of cell surface receptors, expressed by leukocytes, which play roles in host defense. Integrin’s are transmembrane proteins, which bind ligands found in extracellular matrix. Integrin’s: the molecular glue of life. BLAD: White blood cells fail to migrate to the point of infection to destroy invading pathogens. D128G mutation in cattle: Aspartic acid at amino acid 128 changed to glycine in CD18.Understand how amino acid changes can lead to the disorder in dogs (CLAD): Pups with CLAD usually die early in life from multiple severe infections (skin and bone marrow, even when treated with massive doses of antibiotics. Leif Anderson (U of Uppsala, Sweden): CLAD mutation easy to identify because of similarity to human LAD and bovine BLAD. CLAD disorder due to a missense mutation in CD18 gene (Cys36Ser) in Irish Settlers (Carrier: GC, Normal: GG). Affected animals die because of extreme susceptibility to infections, caused by an inability of white blood cells (leukocytes) to pass from the bloodstream into infected tissue. Lack of a membrane glycoprotein called the leukocyte integrin beta-2 subunitor CD18. Inheritance is autosomal recessive.The use of the RFLP test to diagnose and eliminate BLAD: In Holsteins, AI increased the frequency of the defective allele. 1988: 28% of young bulls were found to be BLAD carriers. 1993: BLAD carrier bulls for AI sire proof evaluations were eliminated using genetic testing.How does the mutation affect expression of CD18? D128G mutation in cattle: Aspartic acid at amino acid128 changed to glycine in CD18. Mutation changes the leukocyte so it cannot attach to the vessel wall and reach infected tissues. Mutation occurs near the center of 26 consecutive amino acids that are identical in normal bovine, human and murine CD18.Lecture 3 (January 28)Deficiency of uridine monophosphate synthetase (DUMPS)Understand the difference between the heterozygotes and homozygotes for DUMPS mutation: Autosomal recessive disorder that results in early embryonic mortality at day 40 of gestation. No living animals that are homozygous for the mutated allele. Heterozygous animals live normally; have 50% of normal enzyme activity, and increased orotic acid in urine and milk. Heterozygous x heterozygous mating’s require ~3.1 services per calving, compared to ~2.0 for normal x normal mating’s. All carriers are descendants of Skokie Sensation Ned born in 1957. Testing for DUMPS has greatly reduced the frequency of heterozygous sires and of homozygous recessive embryos.Orotic acid Uridine monophosphate Synthetase (UMPS) Uridine monophosphate monomer in RNADetection of DUMPS mutation: Measure the UMPS activity in erythrocytes. Heterozygotes show half the normal activity. Enzyme activity may be affected by different factors... RNA was extracted from livers of carriers and normal individualsReverse transcription into cDNASequence of RT-PCR productsHeterozygous individuals: C/T in codon 405; arginine CGA to stop codon TGAResult: truncated