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Cloning, Organism: The formation of one or more genetically identical organisms derived by vegetative reproduction from a single cell. The source nuclear material can be embryo-derived, fetus-derived, or taken from an adult somatic cell.

Molecular Cloning

JoVE 5074

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called ligation. The enzyme that catalyzes the ligation reaction is known as ligase. This video explains the major methods that are combined, in tandem, to comprise the overall molecular cloning procedure. Critical aspects of molecular cloning are discussed, such as the need for molecular cloning strategy and how to keep track of transformed bacterial colonies. Verification steps, such as checking purified plasmid for the presence of insert with restrictions digests and sequencing are also mentioned.


 Basic Methods in Cellular and Molecular Biology

Recombineering and Gene Targeting

JoVE 5553

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based on stretches of similar or identical sequences. Together with gene targeting, which takes advantage of endogenous HR to alter an organism’s genome at a specific loci, HR-based cloning techniques have greatly improved the speed and efficacy of high-throughput genetic engineering.In this video, we introduce the principles of HR, as well as the basic components required to perform a recombineering experiment, including recombination-competent organisms and genomic libraries such as bacterial artificial chromosomes (BAC). We then walk through a protocol that uses recombineering to generate a gene-targeting vector that can ultimately be transfected into embryonic stem cells to generate a transgenic animal. Finally, several applications that highlight the utility and variety of recombineering techniques wi


 Genetics

Yeast Transformation and Cloning

JoVE 5083

S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures.

This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast plasmids will be presented, along with the procedure required to prepare yeast cells to incorporate new plasmids. The presentation also includes a step-by-step protocol for the lithium acetate method of yeast transformation. Finally, examples of the many applications of this essential technique will be provided.


 Biology I

An Overview of Genetic Engineering

JoVE 5552

Genetic engineering – the process of purposefully altering an organism’s DNA – has been used to create powerful research tools and model organisms, and has also seen many agricultural applications. However, in order to engineer traits to tackle complex agricultural problems such as stress tolerance, or to realize the promise of gene therapy for treating human diseases, further advances in the field are still needed. Important considerations include the safe and efficient delivery of genetic constructs into cells or organisms, and the establishment of the desired modification in an organism’s genome with the least “off-target” effects. JoVE’s Overview of Genetic Engineering will present a history of the field, highlighting the discoveries that confirmed DNA as the genetic material and led to the development of tools to modify DNA. Key questions that must be answered in order to improve the process of genetic engineering will then be introduced, along with various tools used by genetic engineers. Finally, we will survey several applications demonstrating the types of experimental questions and strategies in the field today.


 Genetics

An Introduction to Saccharomyces cerevisiae

JoVE 5081

Saccharomyces cerevisiae (commonly known as baker’s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans). This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer’s and Parkinson’s diseases.


 Biology I

RNAi in C. elegans

JoVE 5105

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.


 Biology I

CRISPR Guide RNA Cloning for Mammalian Systems

1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute

JoVE 57998


 Genetics

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

1Department of Health Sciences, Università del Piemonte Orientale & IRCAD, Novara, Italy, 2Institute of Biomedical Technologies, National Research Council, Segrate, Milan, Italy, 3Institute of Biomedical Technologies, National Research Council, Bari, Italy, 4Department of Life Sciences, University of Trieste, Italy, 5Institute of Genetic and Biomedical Research, National Research Council, Rozzano, Milan, Italy, 6Humanitas Clinical and Research Center, Rozzano, Milan, Italy

JoVE 56981


 Biology

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

1Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona, 2Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, 3MTA-DE “Lendulet” Immunogenomics Research Group, University of Debrecen

JoVE 53978


 Developmental Biology

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 3Crop and Environment Sciences, Harper Adams University

JoVE 55534


 Immunology and Infection

Competitive Genomic Screens of Barcoded Yeast Libraries

1Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Donnelly Sequencing Centre, University of Toronto, 4Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, 5Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, 6Department of Pharmaceutical Sciences, University of Toronto

JoVE 2864


 Biology

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

1Beijing Key Laboratory of New Technology in Agricultural Application, National Demonstration Center for Experimental Plant Production Education, Beijing University of Agriculture, 2College of Plant Protection, State Key Laboratory of Agrobiotechnology, Ministry of Agriculture Key Laboratory of Pest Monitoring and Green Management, China Agricultural University

JoVE 57675


 Environment

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