Prof. Fahd M. Nasr. Faculty of Sciences Lebanese University Beirut, Lebanon.

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1 Prof. Fahd M. Nasr Faculty of Sciences Lebanese University Beirut, Lebanon

2 Biol328 - B3212 Molecular Biotechnology Partial Exam

3 Question I Explain the procedure for the production of transgenic animals by Somatic Cell Nuclear Transfer (SCNT) or cloning. Three approaches have been devised to achieve nuclear transplantation. What are they? What is a cytoplast? Flow cytometry is used to isolate cytoplasts, how does it work? Is the cloned animal transgenic or non-transgenic? Justify your answer.

4 Somatic Cell Nuclear Transfer The egg is enucleated a cytoplast A diploid nucleus is isolated from an adult cell injected into the cytoplast A diploid cell a zygote Electrical pulse the zygote start to divide a blastocyst a foster mother Nucleus reprogrammed a whole normal organism

5 Somatic Cell Nuclear Transfer (SCNT)

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7 Three approaches used Hand-made cloning approach designed by Vajta et al. (2001).This technique requires no micromanipulator. Nuclear injection strategy as imagined by Spemann and used for the first time by King and Briggs (1952) to clone an amphibian. A cell fusion approach used by several scientists e.g. by Wilmut to clone Dolly the lamb in 1996.

8 Hand-made cloning Nuclear injection Cell fusion approach

9 Flow Cytometry (FCM) or FACS A method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific fluorescent characteristics of each cell The system is adjusted so that there is no more than one cell per droplet

10 Flow Cytometry FACS = Fluorescence-activated cell sorter

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12 Transgenic or not If the nuclei donor cells have been genetically modified transgenic clone If not non-transgenic

13 Question II What are the main types of transposable elements (TEs)? To which type belongs the "Sleeping Beauty" (SB) transposon? Describe the SB transposon vector systems. SB-based tools can be used in gene delivery and gene therapy. Explain how.

14 TEs come in two types The "copy and paste" retrotransposons RNA copy RT to DNA integrated in the genome The "cut and paste" TEs direct excision from DNA insertion elsewhere in the genome

15 Structural features of SB transposase The Nt DNA-recognition domain binding to the DR sequences two paired boxes are labeled PAI and RED A nuclear localization sequence (NLS) A DDE domain catalyzes the cut-and-paste set of reactions transposition found in many transposase and recombinase enzymes

16 The SB transposon system Composed A Sleeping Beauty (SB) transposase and a transposon insert specific sequences of DNA into genomes of vertebrate animals DNA transposons translocate from one DNA site to another in a simple, cut-andpaste manner

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20 Question III Discuss briefly how the mammary gland could be used as a bioreactor for the production of commercial products? Give an example. How does "animal bioreactor" compare with "molecular farming"?

21 How may mammary tissue be used to produce recombinant protein? Promoter of a major milk protein recombinant drug gene Clone the cdna is under the control of the WAP gene promoter Expressed in the mammary tissue only!

22 Example "Genie" The first genetically engineered animal to produce a human protein drug (human protein C) in her milk Blood protein Functions to control blood clotting Some individuals have inborn deficiency Require exogenous Protein C

23 Question IV RNA interference (RNAi) is a mechanism for the suppression of gene expression in a homology dependent manner. What are sirnas? How are they produced? What is the difference between the cleavage and the bypass pathways of gene silencing by RNAi?

24 sirnas Small dsrna of 21-23bp with 2 nucleotides overhangs either side of the RNA duplex Long dsrna produced in several ways Transcription of inverted repeat Transcription of sense and antisense strands Transcription of sense and antisense genes Long dsrna sirna denaturation guide binds target mrna block expression

25 RNAi Post Transcription Gene Silencing Two different pathways sirna (Small interfering RNA) cleavage mechanism strna or mirna (Small temporal RNA or micro RNA) bypass mechanism

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27 Question V The advanced modern biotechnology claims to have all the tools and strategies to "heal, fuel, and feed" the world. How is that possible? (you may list some strategies pertaining to each of the three objectives).

28 Heal, feed and fuel the world Heal the World nature's own toolbox Reducing rates of infectious disease Saving millions of children's lives Tailoring treatments to individuals to minimize health risks and side effects Creating precise tools for disease detection Combating serious illnesses and everyday threats confronting the developing world

29 Recent advances in biotechnology Fuel the World Reducing use of and reliance on petrochemicals Using biofuels to cut greenhouse gas emissions Decreasing water usage and waste generation Feed the World Generating higher crop yields Lowering volumes of agricultural chemicals Developing crops with enhanced nutrition profiles Improving food and crop oil content to help improve cardiovascular health

30 The end