We think you have liked this presentation. If you wish to download it, please recommend it to your friends in any social system. Share buttons are a little bit lower. Thank you!
Presentation is loading. Please wait.
Published byNickolas Weaver
Modified about 1 year ago
BioSci D145 lecture 1 page 1 © copyright Bruce Blumberg All rights reserved BioSci D145 Lecture #8 Bruce Blumberg –4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment) –phone TA – Bassem Shoucri –4351 Nat Sci 2, , 3116 – office hours M 2-4 lectures will be posted on web pages after lecture –http://blumberg.bio.uci.edu/biod145-w2015http://blumberg.bio.uci.edu/biod145-w2015 –http://blumberg-lab.bio.uci.edu/biod145-w2015http://blumberg-lab.bio.uci.edu/biod145-w2015 Term papers due Friday, March 6 by 12 midnight (23:59.59) (-1 point for each day late)
BioSci D145 lecture 1 page 2 © copyright Bruce Blumberg All rights reserved Term paper requirements and scoring Outline - 1 point Actual paper – 5 pages single spaced 1” margins (references not included). Specific aims – 2 points (this should be about 3/4 to one page) –Write a paragraph introducing the topic, state why it is important and what are the gaps in knowledge that you will address. –State a hypothesis to be tested –Enumerate 2-3 specific aims in the form of questions that test your hypothesis. After each one, use a sentence or two to state what you will do to answer these questions –Finish with a paragraph stating what will be the significance of the research assuming that you successfully execute the proposed experiments. –It is very important to state the human health relevance of your research (if you are doing something biomedical) or the broader impacts on advancing the frontiers of knowledge (for something that is not relevant to human health). –This is among the most important parts of any grant application. You have to convince the reviewer here that your work is important and worth funding.
BioSci D145 lecture 1 page 3 © copyright Bruce Blumberg All rights reserved Term paper requirements and scoring Background and Significance – 3 points (about pages) –Briefly summarize what is known about the problem. Not a comprehensive review, just a summary of the important points. –Succinctly state what is not known and why it is important that this research be done Address knowledge gaps Are you addressing something controversial? –talk about the controversy and why your work will address it directly. –In about one paragraph, state what is important about your proposed research and why will accomplishing it benefit the research community and world at large. i.e., what is the potential impact if you are successful Don’t repeat what was said in specific aims exactly but obviously they should be related.
BioSci D145 lecture 1 page 4 © copyright Bruce Blumberg All rights reserved Term paper requirements and scoring Research plan – 4 points (about pages) –In a short paragraph, state what you will do and why it is important. (I know it seems repetitive by now, but reviewers are busy and will be skimming your grant. You need to hit them over the head a few times before they will get your point). –Restate each specific aim from the Specific aims section (one by one) –describe what you will do to address the aim Break into subaims as appropriate State the hypothesis to be tested in each Explain the rationale Describe briefly what approach you will take Discuss what you expect to find Point out any possible problems and alternative approaches –I am mostly concerned with your hypothesis and rationale here. –Not an all-encompassing proposal – 4-5 years by a small team (e.g., your PhD thesis research)
BioSci 145B lecture 6 page 5 © copyright Bruce Blumberg All rights reserved Gene targeting Transgenesis is mostly a gain-of-function technique –Loss-of-function preferred for identifying gene function Targeted gene disruption is very desirable –to understand function of newly identified genes e.g., from genome projects Or gene by gene –produce a mutation and evaluate the requirements for your gene of interest –good to create mouse models for human diseases knockout the same gene disrupted in a human and may be able to understand disease better and develop efficacious treatments excellent review is Müller (1999) Mechanisms of Development 82,
BioSci 145B lecture 6 page 6 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) enabling technology is embryonic stem (ES) cells (or iPS cells) –these can be cultured but retain the ability to colonize the germ line –essential for transmission of engineered mutations –derived from inner cell mass of blastula stage embryos –grown on lethally irradiated “feeder” cells which help to mimic the in vivo condition essential for maintaining stem cell phenotype Also problematic for human ES lines ES cells are very touchy in culture –lose ability to colonize germ line with time –easily infected by “mysterious microorganisms” that inhibit ability to colonize germ line ko labs maintain separate hoods and incubators for ES cell work –ES cells depend critically on the culture conditions maintain an uncommitted, undifferentiated state that allows germ line transmission.
BioSci 145B lecture 6 page 7 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) isolate genomic clones from ES cell library Restriction map –Especially exons/introns Make targeting construct –Want ~5kb genomic regions flanking targeted region –Must disrupt essential exon –Want no functional protein –Verify in cell culture –often useful to fuse reporter gene to the coding region of the protein gene expression can be readily monitored –Insert dominant selectable marker within replacement region –negative selection marker is located outside the region targeted to be replaced Electroporate DNA into ES cells, select colonies resistant to positive selection Integration positive cells then subjected to negative selection –homologous recombinants lose this marker
BioSci 145B lecture 6 page 8 © copyright Bruce Blumberg All rights reserved Gene Targeting (contd) Targeting vector Electroporate into ES cells Recombination Selection identification
BioSci 145B lecture 6 page 9 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) Technique (contd) –homologous recombination is verified by Southern blotting –factors affecting targeting frequency (success) length of homologous regions, more is better. –0.5 kb is minimum length for shortest arm isogenic DNA (ie, from the ES cells) used for targeting construct is best locus targeted. This may result from differences in chromatin structure and accessibility –Expand ES cell colonies
BioSci 145B lecture 6 page 10 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) –Transfer into blastocyst of recipient –Implant into foster mothers (white in this diagram, but actually black) Progeny will be mixed color – brown from ES cells, black from host –Breed mixed color F1 mice with homozygous white mice –Black progeny derive from germ cells harboring the knockout Heterozygous for knockout –Breed these to establish lines and determine effects of homozygous mutations
BioSci 145B lecture 6 page 11 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) problems and pitfalls –incomplete knockouts, ie, protein function is not lost but such weak alleles may be informative –alteration of expression of adjacent genes region removed may contain regulatory elements may remove unintended genes (e.g. on opposite strand) –interference from selection cassette strong promoters driving these may cause phenotypes
BioSci 145B lecture 6 page 12 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) Applications –creating loss-of-function alleles –introducing subtle mutations –chromosome engineering –marking gene with reporter, enabling whole mount detection of expression pattern (knock-in)
Example of a “knock-in” model BioSci D145 lecture 8 page 13 © copyright Bruce Blumberg 2009 All rights reserved
BioSci 145B lecture 6 page 14 © copyright Bruce Blumberg All rights reserved Gene targeting (contd) Applications –creating loss-of-function alleles –introducing subtle mutations –chromosome engineering –marking gene with reporter, enabling whole mount detection of expression pattern (knock-in) advantages –can generate a true loss-of-function alleles –precise control over integration sites –prescreening of ES cells for phenotypes possible –can also “knock in” genes disadvantages –not trivial to set up –may not be possible to study dominant lethal phenotypes –non-specific embryonic lethality is common (~30%) –difficulties related to selection cassette
BioSci D145 lecture 8 page 15 © copyright Bruce Blumberg 2009 All rights reserved Conditional gene targeting Many gene knockouts are embryonic lethal –some of these are appropriate and expected gene activity is required early –others result from failure to form and/or maintain the placenta ~30% of all knockouts Clearly a big obstacle for gene analysis How can this be overcome? –Generate conditional knockouts either in particular tissues or after critical developmental windows pass –Sauer (1998) Methods 14,
BioSci D145 lecture 8 page 16 © copyright Bruce Blumberg 2009 All rights reserved Conditional gene targeting - contd Approach –recombinases perform site-specific excision between recognition sites –FLP system from yeast doesn’t work well –Cre/lox system from bacteriophage P1 P1 is a temperate phage that hops into and out of the bacterial genome recombination requires –34 bp recognition sites locus of crossover x in P1 (loxP) –Cre recombinase if loxP sites are directly repeated then deletions if inverted repeats then inversions result
BioSci D145 lecture 8 page 17 © copyright Bruce Blumberg 2009 All rights reserved Conditional gene targeting (contd) Strategy –Make targeting construct (minimum needed for grant) –homologous recombination, select for loss of DT-A –transfect CRE, select for loss of tk –Southern to select correct event Result called “floxed allele” –inject into blastocysts, select chimeras –establish lines –cross with Cre expressing line and analyze function
BioSci D145 lecture 8 page 18 © copyright Bruce Blumberg 2009 All rights reserved Conditional gene targeting (contd) –Tissue- or stage-specific knockouts from crossing floxed mouse with specific Cre-expressing line –requirement for Cre lines must be well characterized –promoters can’t be leaky Andras Nagy’s database of Cre lines and other knockout resources a/cre_new/index.php a/cre_new/index.php
Figure 5.25 Floxing mice
BioSci D145 lecture 8 page 20 © copyright Bruce Blumberg 2009 All rights reserved Conditional gene targeting (contd) advantages –can target recombination to specific tissues and times –can study genes that are embryonic lethal when disrupted –can use for marker eviction –can study the role of a single gene in many different tissues with a single mouse line –can use for engineering translocations and inversions on chromosomes disadvantages –not trivial to set up, more difficult than std ko but more information possible –requirement for Cre lines must be well characterized regarding site and time of expression promoters can’t be leaky (expressed when/where not intended)
BioSci D145 lecture 8 page 21 © copyright Bruce Blumberg 2009 All rights reserved Other approaches to genome manipulation Transgenic and knockout technology is species dependent (doesn’t work in all species – need ES cell equivalent). How else can we accomplish gene disruption in a targeted way ? –RNAi approaches (Boutros, Luo papers this week) –Nuclease based methods - introduce double-stranded breaks ZFN – zinc finger nucleases TALEN nucleases Meganuclease –CRISPR/Cas – RNA guided nuclease (paper this week) Meganucleases –Based on naturally occurring restriction enzymes with extended DNA binding specificity (relatively limited application) TALEN and ZFN nucleases –Artificial fusion proteins combining an engineered DNA binding domain fused to a nonspecific nuclease domain from FokI restriction enzyme –ZNF – zinc finger repeats –TALE – repeats –Main limitation is the sequence specificity that can be engineered in.
BioSci D145 lecture 8 page 22 © copyright Bruce Blumberg 2009 All rights reserved CRISPR-Cas gene editing CRISPR = clustered, regularly interspaced, short palindromic repeat Sander & Joung (2014) CRIRISPR-Cas systems for editing, regulating and targeting genomes, Nat Biotech 32: –Cas9 – RNA-guided nuclease –Functions as a bacterial immune system. Foreign DNA is incorporated between CRISPR repeat sequences, transcription generates crRNA. –Hybridizes to tracrRNA (also encoded by CRISPR system), guides Cas9 nuclease to the target –Cas9 nuclease introduces double stranded breaks into target –Repair of this break can introduce deletions frameshifts, etc.
BioSci D145 lecture 8 page 23 © copyright Bruce Blumberg 2009 All rights reserved CRISPR-Cas gene editing CRISPR/Cas9 (contd) –For gene targeting, we would fuse the target RNA to the tracrRNA and introduce this into the target cell, embryo, etc together with Cas9 nuclease –Introduces ds breaks at target sequence which may introduce desirable mutations. –Potential issues Can’t specify what happens after ds break (deletion, frameshift, insertion, etc Some question about specificity of the crRNA introduced Some sequence preferences of Cas9 nuclease may limit utility
BioSci D145 lecture 8 page 24 © copyright Bruce Blumberg 2009 All rights reserved CRISPR-Cas gene editing - applications
BioSci D145 lecture 8 page 25 © copyright Bruce Blumberg 2009 All rights reserved Generating phenocopies of mutant alleles How to inactivate endogenous genes in a targeted but general way? –Important new development is RNAi – RNA interference –Observation is that introduction of double- stranded RNAs into cells lead to destruction of corresponding mRNA (if there is one) –Principle is siRNA – small interfering RNAs –These generate small single stranded RNAs that target mRNAs for destruction by –RISC – RNA interference silencing complex –First applied in C. elegans where it works extremely well Can introduce siRNA into cells even by feeding to the worms! Works very well in Drosophila variably in mammalian cells Poorly in Xenopus – Why? Xenopus has an endogenous helicase
BioSci D145 lecture 9 page 26 © copyright Bruce Blumberg All rights reserved RNAi (contd) Dicer complex generates short duplexes from dsRNA in the cell –Important to have 2-nt overhangs siRNAs are generated from these fragments –Antisense strand binds to mRNA and this recruits the RISC - RNAi silencing complex –Complex leads to mRNA cleavage and destruction Two important reviews to read –McManus and Sharp (2002) Nature Reviews Genetics 3, –Dykxhoorn et al. (2003) Nature Reviews, Molecular Cellular Biology 4,
BioSci D145 lecture 9 page 27 © copyright Bruce Blumberg All rights reserved RNAi (contd) Micro RNAs are small cellular RNAs that previously lacked any known function –Always form a hairpin structure with mismatches in stem Micro RNAs direct gene silencing via translational repression –(miRNAs) are mismatched duplexes that dicer processes into stRNAs (small temporal RNAs) –Use same cellular complex as siRNAs –Perfect matches -> target cleavage –Imperfect matches -> translational repression of target Two important papers –Giraldez et al (2005) Science 308, (microRNAs regulate brain morphogenesis) –Lecellier et al (2005) Science 308, (microRNA mediates antiviral defenses in human cells)
BioSci D145 lecture 9 page 28 © copyright Bruce Blumberg All rights reserved RNAi (contd) Parallels between siRNA and miRNA-directed RNAi
BioSci D145 lecture 9 page 29 © copyright Bruce Blumberg All rights reserved RNAi (contd) Ways to generate short RNAs that silence gene expression in vitro –a) chemical synthesis of siRNA, introduce into cell –b) synthesize long dsRNA, use dicer to chop into siRNA –c) introduce perfect duplex hairpin, dicer generates siRNA –d) make miRNA based hairpin, dicer generates silencing RNA Introduce into cells or organism by microinjection, transfection, etc. –Expression is transient, loss of function ALWAYS partial –can only generate phenotypes for a short time after introduction
BioSci D145 lecture 8 page 30 © copyright Bruce Blumberg 2009 All rights reserved RNAi (contd) Ways to generate short silencing RNAs in vivo – need continuing expression to generate stable phenotype –a) produce long hairpin from pol II promoter, let dicer make siRNA –b) produce two transcripts from pol III promoter, let anneal in cells –c) produce a short hairpin from pol III promoter (or viral vector), let dicer generate siRNAs –d) produce imperfect hairpin from pol II promoter, let dicer generate miRNAs that direct gene silencing
BioSci D145 lecture 9 page 31 © copyright Bruce Blumberg All rights reserved RNAi (contd) RNAi for whole genome functional analysis –First generate library of constructs that generates siRNA or stRNA Or synthesize a complete library of siRNAs –Introduce these into cells, embyos (fly, frog, mouse) or animals (C. elegans, plants) For C. elegans, make the library in E. coli and simply feed bacteria to worms Must microinject or transfect with other animals –Evaluate phenotypes IMPORTANT! –RNAi is inherently transient unless you are expressing the siRNAs from a stably integrated plasmid –RNAi is a knock-down (not knock-out) method.
BioSci D145 lecture 8 page 32 © copyright Bruce Blumberg 2009 All rights reserved Phenotypic rescue by RNAi – synthetic lethal and related approaches How can we find other members of pathways we already know something about? –Or, how can we find drugs that act on a pathway to kill cells (e.g., cancer cells?) –Synthetic lethal is one relatively new and promising approach 2 mutations are synthetic lethal if either single mutation is viable but the double mutant is lethal
BioSci D145 lecture 8 page 33 © copyright Bruce Blumberg 2009 All rights reserved Phenotypic rescue by RNAi – synthetic lethal and related approaches How can we find other members of pathways we already know something about? –In cancer screening, what if we combine a mutation and a drug to find a combination that increases the kill rate, or reveals a phenotype that is similar to the total loss of function? Could find novel drug targets (pathways that kill cells in presence of siRNA+drug (usually sublethal amount of drug) Can find genes that are targeted by drugs – pathway analysis Could find new biomarkers that are required for cell viability following drug treatment
BioSci D145 lecture 8 page 34 © copyright Bruce Blumberg 2009 All rights reserved Mohr et al., 2010, Genomic Screening with RNAi: Results and Challenges. Annual Review of Biochemistry, 29: 37-64
BioSci D145 lecture 8 page 35 © copyright Bruce Blumberg 2009 All rights reserved Genome wide analysis of gene function How to mutate all genes in a given genome? –Easy with microbial genomes – can mutate all yeast genes by homologous recombination –Recombine in selectable marker –Propagate strain and analyze phenotypes Target gene Selectable marker (antibiotic resistance) Homology region Unique oligonucleotide “barcodes” for PCR
BioSci D145 lecture 8 page 36 © copyright Bruce Blumberg 2009 All rights reserved Genome wide analysis of gene function (contd) How about gene targeting in other organisms –With more complex genomes and more genes? –Huge undertaking to specifically target 30K+ genes in mammalian cells Difficulty Expense Inability to target all possible loci –Some efforts to make mouse collection Lexicon Genetics has a collection of ES cells –Drosophila collection as well –Driving force behind these efforts is Genome annotation Drug target discovery (Lexicon) Functional analysis
BioSci D145 lecture 8 page 37 © copyright Bruce Blumberg 2009 All rights reserved Figure 5.18 The three major types of mutagen
BioSci D145 lecture 8 page 38 © copyright Bruce Blumberg 2009 All rights reserved Genome wide analysis of gene function (contd) Main method for gene targeting in more complex organisms is random insertional mutagenesis –Transposon mutagenesis Bacteria – Tn transposons Yeast - Ty transposons Drosophila - P- elements Vertebrates - Sleeping Beauty transposons –Viral infection Typically retroviruses – host range selectivity is obstacle –Gene or enhancer trapping – modified viruses or transposons
BioSci D145 lecture 8 page 39 © copyright Bruce Blumberg 2009 All rights reserved Insertional mutagenesis - Gene trapping – enhancer trip enhancer trap is designed to bring inserted reporter gene under the control of local regulatory sequences –put a reporter gene adjacent to a weak promoter (enhancer-less), e.g. a retrovirus with enhancers removed from the LTRs –may or may not disrupt expression –Hopkins zebrafish group used unmodified virus viruses and transposable elements can deliver DNA to random locations –can disrupt gene function –put inserted gene under the control of adjacent regulatory sequences –BOTH
BioSci D145 lecture 8 page 40 © copyright Bruce Blumberg 2009 All rights reserved Insertional mutagenesis - Gene trapping –enhancer trap (contd) Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like. Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K. Development 2008 Jan;135(1):
BioSci D145 lecture 8 page 41 © copyright Bruce Blumberg 2009 All rights reserved Insertional mutagenesis - Gene trapping –enhancer trap (contd) stopped here 2015 enhancer trap (contd) –expression only when integrate into an active transcription unit reporter expression duplicates the temporal and spatial pattern of the endogenous gene –reporters used -galalactosidase was the most widely used reporter GFP is now popular -lactamase is seeing increasing use –advantages relatively simple to perform active promoters frequently targeted, perhaps due to open chromatin –Disadvantages Inactive promoters probably not targeted insertional mutagenesis not the goal, and not frequent –overall frequency is not that high relies on transposon or retroviruses to get insertion –may not be available for all systems, requires transgenesis or good viral vectors
BioSci D145 lecture 8 page 42 © copyright Bruce Blumberg 2009 All rights reserved Insertional mutagenesis – Gene trapping (contd) expressed gene trap (many variations possible) –goal -> ablate expression of endogenous gene, replace with transgene –Make insertion construct – reporter, selection, polyA sites No promoter but has a splice-acceptor sequence 5’ of reporter Can only be expressed if spliced into an endogenous mRNA –Transfer into embryonic cells, generate a library of insertional mutagens Mouse, Drospophila, zebrafish, frog –reporter expression duplicates the temporal and spatial pattern of the endogenous gene
BioSci D145 lecture 8 page 43 © copyright Bruce Blumberg 2009 All rights reserved Insertional mutagenesis - Gene trapping (contd) Expressed gene trapping (contd) –advantages insertional mutagen –gives information about expression patterns –can be made homozygous to generate phenotypes higher efficiency than original trapping methods selectable markers allow identification of mutants –many fewer to screen –dual selection strategies possible –disadvantages overall frequency is still not that high frequency of integration into transcription unit is not high either relies on transposon or retroviruses to get insertion –may not be available in your favorite system. –Uses Insertional mutagenesis Marking genes to identify interesting ones Gene cloning
BioSci D145 Lecture #8 Bruce Blumberg –4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment) –phone TA – Ron Leavitt.
Control of Gene Expression. Ways to study protein function by manipulating gene expression Mutations –Naturally occurring, including human and animal.
Next lecture:techniques used to study the role of genes in develpoment Random genetics followed by screening Targeted mutagenesis (gene knockout) Transgenic.
Transfection. What is transfection? Broadly defined, transfection is the process of artificially introducing nucleic acids (DNA or RNA) into cells, utilizing.
BioSci 145A lecture 12 page 1 © copyright Bruce Blumberg All rights reserved Lecture /14/2001 Transcription factors I Topics we will cover.
2 March, 2005 Chapter 12 Mutational dissection Normal gene Altered gene with altered phenotype mutagenesis.
Advantages of C. elegans: 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction 4. transparent 5. only ~1000 cells 6. laser ablation 7. complete.
DNA Chips Attach DNA to tiny spots on glass slides (i.e., chip). Hybridize fluorescently-labeled DNA probes to chip. Detect hybridization to different.
Transgenic Mice (not nec. KO) 1. How to get the DNA in? 2. Where is it, once inside?
RNA-ligand interactions and control of gene expression.
MBP1007/ Nucleic Acids A functional mRNA: The cytoplasmic story Objectives (1) To discuss the iNUTS and iBOLTS of how mRNAs function in the cytoplasm.
Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Forward genetics and reverse genetics Forward genetics: from phenotype to gene sequence Reverse genetics : from gene sequence to phenotype Gene Mutation.
Synthetic biology Genome engineering Chris Yellman, U. Texas CSSB.
Understanding genetic tools in haematology research.
Recombinant DNA & Biotechnology. Recombinant DNA recombinant DNA molecules contain DNA from different organisms –any two DNAs are joined by DNA ligase.
Mouse as a Model Organism Tuesday, February 7, 2012.
Transgenic Mouse: Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from.
15 March 2016 Today’s Title: CW: Introduction to genetic engineering Learning Question: what is genetic engineering?
General Microbiology (Micr300) Lecture 11 Biotechnology (Text Chapters: ; )
Vectors for RNAi. Phenomena first observed in petunia Attempted to overexpress chalone synthase (anthrocyanin pigment gene) in petunia. (trying to darken.
18. Transgenic Models. Approaches Used in the Analysis of Mammalian Development Observations during embryogenesis Phenotypic analysis of developmental.
C. Elegans. Purpose of the Lab To learn about DNA Inject DNA into living organisms in an attempt to have the offspring express the traits Stop gene silencing.
Chap. 5 Problem 1 Recessive mutations must be present in two copies (homozygous) in diploid organisms to show a phenotype (Fig. 5.2). These mutations show.
Reverse Genetics in Drosophila I. P elements in reverse genetics A. P element insertional mutagenesis projects B. Using P elements to make mutations II.
General Genetics Dr. Attya Bhatti. Genetic Engineering Also known as Gene manipulation Genetic modifications recombinant DNA technology, New Genetics.
RNA interference Definition: RNA interference (RNAi) is a mechanism where the presence of certain fragments.
Advanced Microbial Physiology Lecture 1 Minimum Bacterial Genome.
Lecture 5 Recombinant DNA Technology Cloning Vectors Gene Libraries Clone Identification and Characterization Reading: Chapter 9 Molecular Biology syllabus.
BioSci 145B lecture 9 page 1 © copyright Bruce Blumberg All rights reserved BioSci 145B Lecture #9 5/31/2005 Bruce Blumberg –2113E McGaugh Hall -
Recombinant DNA Technology CHMI 4226 E Week of April 30, 2009 Functional genomics Transgenic mice Knock-out mice.
Transfection-Transfer of non-viral genetic material into eukaryotic cells. Infection/ Transduction- Transfer of viral genetic material into cells.
DNA Technology and Genomics Ch. 20. Genetic engineering Genetic engineering: the direct manipulation of genes for “practical” purposes When genes from.
Key Area : Genetic Control of Metabolism in Micro-organisms Unit 2: Metabolism and Survival.
Genetics in the real world: Developing a new genetic system in bacteria Abigail Salyers
Molecular Techniques. Recombinant DNA Technology Provides ability to isolate, sequence, and manipulate individual genes derived from any type of cell.
Gene tagging To study the gene and its functions : -Forward genetics: Involves random mutagenesis followed by screening to recover mutants showing impairment.
9.1 Manipulating DNA. KEY CONCEPT Biotechnology relies on cutting DNA at specific places.
Learning question: What are the applications of genetic engineering? Title: Genetic Techniques 1 Starter: Can you think of any real life applications of.
Recombinant DNA Technology Site directed mutagenesis Genetics vs. Reverse Genetics Gene expression in bacteria and viruses Gene expression in yeast Genetic.
Biotechnology Techniques in Developmental Biology Ch. 5 - Gilbert pp
RNAi. What is RNAi? RNA-based mechanisms of gene silencing. These siRNAs are bound by a protein-RNA complex called the RNA-induced silencing complex (RISC)
Transfection Key words: Transient transfection, Stable transfection, transfection methods, vector, plasmid, origin of replication, reporter gene/ protein,
Chapter 9 – DNA-Based Information Technologies Recombinant DNA molecules are constructed with DNA from different sources Recombinant DNA molecules are.
Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 9 Biotechnology and Recombinant DNA.
An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells Scott M. Hammond, Emily Bernstein, David Beach, and Gregory J.
Chapter 12 Lecture Outline Molecular Techniques and Biotechnology.
Problem 1. You screen two libraries- cDNA; genomic 2. Clones are isolated having homology to PSY- 10 clones from each library 3. These are subcloned into.
Mutagenesis Methods Lily Peterson April 5 th, 2010.
© 2017 SlidePlayer.com Inc. All rights reserved.