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Homologous Recombination: An exchange of DNA between matching or similar sequences.

In-vitro Mutagenesis

JoVE 10813

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

Genes can be randomly knocked out, or specific genes can be targeted. To knock out a particular gene, an engineered piece of DNA called a targeting vector is used to replace the normal gene, thereby inactivating it. Targeting vectors have sequences on each end that are identical—or homologous— to the sequences flanking each side of the gene of interest. These homologous sequences allow the targeting vector to replace the gene through homologous recombination—a process that occurs naturally between DNA with similar sequences during meiosis. The targeting vector is introduced into mouse embryonic stem cells in culture, using methods such as electroporation—use of electric pulses to temporarily create pores in the cell membrane. Typically, to identify cells where the vector has properly replaced the gene, it is designed to include a positive selection marker—such as the gene for neomycin resistance (NeoR)—between the homologous regions; and a negative selection marker—such as th

 Core: Biotechnology

Crossing Over

JoVE 10769

Unlike mitosis, meiosis aims for genetic diversity in its creation of haploid gametes. Dividing germ cells first begin this process in prophase I, where each chromosome—replicated in S phase—is now composed of two sister chromatids (identical copies) joined centrally.

The homologous pairs of sister chromosomes—one from the maternal and one from the paternal genome—then begin to align alongside each other lengthwise, matching corresponding DNA positions in a process called synapsis. In order to hold the homologs together, a protein complex—the synaptonemal complex—forms. The synaptonemal complex facilitates the exchange of corresponding random pieces of DNA between non-sister chromatids, yielding new combinations of alleles via homologous recombination. As the synaptonemal complex begins to dissolve, X-shaped structures hold the homologous chromosomes together until recombination is completed. The structures—called chiasmata—mark the areas where crossover of genetic information occurred.

 Core: Meiosis

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

1Institute for Microbiology, Heinrich-Heine University Düsseldorf, 2Bioeconomy Science Center (BioSC), 3Department of Genetics, Institute of Applied Biosciences, Karlsruhe Institute of Technology, 4Cluster of Excellence in Plant Sciences (CEPLAS), Heinrich-Heine University Düsseldorf

JoVE 54522

 Genetics

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…

 Genetics

Genome Editing

JoVE 5554

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target…

 Genetics

What is Genetic Engineering?

JoVE 10806

Genetic engineering is the process of modifying an organism’s DNA to introduce new, desirable traits. Many organisms, from bacteria to plants and animals, have been genetically modified for academic, medical, agricultural, and industrial purposes. While genetic engineering has definite benefits, ethical concerns surround modifying humans and our food supply.

Genetic engineering is possible because the genetic code—the way information is encoded by DNA—and the structure of DNA are universal among all life forms. As a result, an organism’s genetic code may be modified in several ways. The nucleotide sequence may be selectively edited by using techniques such as the CRISPR/Cas9 system. Known as the "molecular scissors," the CRISPR/Cas9 system is an innate, prokaryotic immune response that has been co-opted for editing genetic information. A gene may also be removed from an organism to create a “knockout,” or introduced to create a “knockin,” through a process called gene targeting. This method relies on homologous recombination—genetic exchange between DNA molecules that share an extended region with similar sequences—to modify an endogenous gene. Scientists can also insert a gene from one organism into the genome of another, resulting in a transgenic organism. Generally, DNA

 Core: Biotechnology

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…

 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…

 Biology I

Genetic Engineering of Model Organisms

JoVE 5327

Transgenesis, or the use of genetic engineering to alter gene expression, is widely used in the field of developmental biology. Scientists use a number of approaches to alter the function of genes to understand their roles in developmental processes. This includes replacement of a gene with a nonfunctional copy, or adding a visualizable tag to a gene that allows the resultant fusion protein to …

 Developmental Biology

An Introduction to Drosophila melanogaster

JoVE 5082

Drosophila melanogaster, also known as the fruit fly, is a powerful model organism widely used in biological research that has made significant contributions to the greater scientific community over the last century. First, this video introduces the fruit fly as an organism, including its physical characteristics, life cycle, environment, and diet. Next, the reasons why fruit flies…

 Biology I

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

1Irell & Manella Graduate School of Biological Sciences, 2Department of Molecular and Cellular Biology, City of Hope Comprehensive Cancer Center and Beckman Research Institute, 3Department of Biochemistry and Molecular Biology, University of Southern California, Norris Comprehensive Cancer Center

JoVE 3150

 Biology

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 2College of Life Sciences, University of Chinese Academy of Sciences, 3School of Life Science and Technology, Shanghai Tech University, 4Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

JoVE 56844

 Genetics

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

1Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 2Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 3Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 4Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, 5Biological and Biomedical Sciences Program, University of North Carolina School of Medicine, 6Department of Radiation Oncology, Emory University School of Medicine, 7Department of Neurology, Neurosciences Center, University of North Carolina School of Medicine

JoVE 51763

 Neuroscience

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

1MRC Laboratory of Molecular Biology, 2Department of Molecular and Cell Biology, University of Connecticut, 3Cancer Research UK Beatson Institute Glasgow, 4MRC Laboratory for Molecular Cell Biology, University College London, 5Department of Cell and Developmental Biology, University College London

JoVE 58981

 Genetics

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

1Department of Biological Chemistry, School of Medicine, University of California, Irvine, 2Beckman Laser Institute and Medical Clinic, University of California, Irvine, 3Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, 4Department of Biomedical Engineering and Surgery, University of California, Irvine

JoVE 56213

 Genetics

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

1Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, 2Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, 3Department of Pathology, UT Health Science Center at San Antonio, 4Department of Microbiology, UT Health Science Center at San Antonio, 5Cancer Therapy and Research Center, UT Health Science Center at San Antonio

JoVE 50752

 Immunology and Infection

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

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan

JoVE 56275

 Genetics
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