MCDB 2150 1st Edition Lecture 21Outline of Last Lecture - Recombinant DNA technologyo How to clone a geneOutline of Current Lecture - Transgenic organismso Transgenes- The “knockout” mouseThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Biotechnology – Transgenic animalscDNA = only contains the genes that are expressed in an organism or tissuecomes from a collection of all mature mRNAs, then reverse transcriptase is used to create the cDNA library (without non-coding regions)Transgenic organisms = contain recombinant DNA in their genomeTransgene = a cloned gene that is transferred from one species to another- ^^must be integrated into a chromosome to become stable- integration:o recombination techniqueso random insertion into a “nick” in the DNAIntroducing transgenes:- gene replacement: cloned gene replaces chromosomal copy by homologous recombination- gene knockout: chromosomal copy replaced with inactive mutant- gene addition: cloned gene integrates at a different site; both copies (original & transgene) are present- Must be done at an early embryonic stage so all cells have recombinant DNAThe transgenic “knockout mouse”- Model for human disease (study of homologs – evolutionarily similar genes)- Most useful for diseases caused by a single gene- 1 – introduce a non-functional copy of the gene by homologous recombination (only occurs in regions where the sequence is similar)o RARE – selection is criticalo Often use NeoR gene (where non-functional gene goes) because this is an anti-toxin gene Grow the animals in the presence of the toxinso BUT – could be random integration ratherthan homologous recom. SOOO…. Use a TK gene as a negative selector (organisms with this gene cannot grow in the presence of gancyclovir) If homologous recombination = only the TK gene or the NeoR gets inserted (not usually
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