Key Technologies Flashcards
What are natural means for introducing genes to cells?
Bacterial transformation - uptake of environmental DNA
Bacterial conjugation - transfer of chromosomal DNA (F factor) between bacteria
Bacteriophage transduction - phage my be lytic, lysogenic or both
What are some artificial means for introducing genes?
Chemical methods - calcium chloride for transformation of E. coli, calcium phosphate for animal cells
Electroporation - produces transient pores, efficient
Biolistics (plants)
Microinjection into egg nuclei
How can we do genetic selection?
DNA can have drug resistance/reporter gene (GFP)
In some cases only small fraction of cells uptake DNA - selected/screened
Introduced DNA can exist extrachromosomally as episome/integrated into chromosomal DNA via recombination
Why are plasmids good vectors?
Direct their own replication and additional factors ensuring the copies get separated into the daughter cells during cell division - not lost during binary fission
Eg. toxin-antitoxin system
How can genes be introduced by transposition?
Transposition is essentially random integration by transposase encoded within transposon
Breaks target DNA and integrates via ends of transposon
Can be replicative/non-replicative
Naturally introduce new genes - vector between inverted terminal repeats
How else are transposons useful?
Can be used as mutagenic agents to disrupt genes and provide a tag to detect it
How does homologous recombination work?
Occurs between regions of homology (quite long)
Involves RecA and RecBCD (E. coli)
Results in integration of incoming DNA, can modify chromosomal sequence
How does non-homologous recombination work?
Doesn’t require homology but can be directed by regions of micro-homology
Major DNA integration method for plants and animals
How can homologous recombination (HR) be used for allele replacement?
Introduced DNA, HR with chromosome/plasmids, Rec-A mediated recombination
2-step method = with plasmid containing homology to genome and +ve and -ve selection markers
Rec-A mediated HR requires pretty large regions of homology
What is Red-mediated homologous recombination?
HR is mediated by RecA pathway and used to make precise modifications - eg introduce gene into BAC - BUT requires large sequences of homology
Alternative = phage lambda Red recombination system - shorter regions of homologous sequence - noe RecA/BC
RecET system from rac prophage similar
Red operon of phage lambda: alpha = endonuclease, beta = annealing protein, and gamma = inhibitor of exonuclease RecBC
How does Red-mediated recombination work?
dsDNA degraded by 5’ to 3’ Red-alpha exonuclease to create ss region
ss coated with Red-beta binding protein
Works by ss annealing, not strand invasion
No need for ATP and only needs short homology arms
DNA target must be replicating
What is Red-mediated ‘recombineering?
Red-mediated recombination exploited for making DNA designed rearrangements in E. coli
Short homologous sequences added by PCR and linear DNA transfected
Alpha, beta, and gamma proteins expressed in E. coli under inducible control
No enzymes required other that PCR polymerase
Rapid and flexible methodfor engineering DNA in E. coli or other bacteria
How do you make linear DNA with homologous ends?
PCR used to make homologous ds linear DNA
3’ ends of PCR primers complimentary to selection marker
5’ ends of primers specify sequence homologous to region in target DNA
Oligonucleotide synthesis allows 5’ ends to be made up to 50-60 nucleotides long for efficient Red-mediated recombination
How does Red-mediated recombineering work?
Lambda Red recombination genes transferred into low copy no. plasmid
Plasmid introduced into E. coli
Linear ‘donor’ DNA made in vitro by PCR and introduced into E. coli
Target DNA incorporates PCR linear and confers drug resistance
How does recombineering with oligonucleotides work?
Recombination with ssDNA (oligonucleotides) very efficient
Bias in efficiency depending on direction of replication fork
Only needs Red-beta annealing protein
Red-beta binds oligonucleotide and anneals it to lagging strand gap in replication fork alongside Okazaki fragments
Can be designed to create diff types of mutation (mismatch, insertion, deletion)
What is MAGE?
Multiplex Automated Genome Engineering
Enables rapid and continuous generation of sequence diversity at many targeted chromosomal locations across a large population of cells through repeated synthetic DNA introduction
Results in rapid evolution of cell population to create strains with desired trait
What is CAGE?
Conjugative Assembly Genome Engineering
Applying conjugation to transfer defined regions of the bacterial chromosome into a recipient strain
oriT derived from F plasmid inserted into desired point on donor chromosome to initiate chromosome transfer; oriT has kanamycin resistance; conjugative functions encoded on plasmid
What is genome editing?
DNA is inserted, replaced, or removed from a genome using artificially engineered endonucleases
What are the two methods of repairing a ds break and when are they most used?
Homologous recombination (HR) - mostly used in bacteria and yeast Non-Homologous End Joining (NHEJ) - mostly used in animal and plant cells
What is a consequence of NHEJ?
Mutagenic as randomly inserts/deletes several base pairs at break site (indels)
How efficient is gene targeting in yeast?
Targeted genome modification occurs 100% of selected cells
Double strand break in the plasmid makes the outcome much more efficient (~100-fold)
How efficient is gene targeting in mammalian cells?
Double strand break also increases frequency of homologous integration but still random integration
What are some problems with DNA injection into egg nuclei?
All integrations occur by non-homologous recombination even if sequence is homologous
Originally could only be used for additive transgenesis
How is plasmid DNA made?
DNA linearised and plasmid backbone sequence removed by restriction enzyme before it’s injected into pronucleus
Contains transgene coding sequence linked to appropriate transcriptional regulatory sequence
