BCH3031 Lecture Notes - Lecture 18: Genome Project, Gene Knockout, Mass Spectrometry
25 May 2018
School
Department
Course
Professor
Ian Smyth
Lecture 18 – Large Scale Approaches to Functional Genetics
• Functional genetics
o A field of molecular biology that attempts to make use of the vast
wealth of data produced by genomic projects (such as genome
sequencing projects) to describe gene (and protein) functions and
interactions
• Genetics
o A discipline of biology, is the science of heredity and variation in
living organisms
• Functional genetics
o The study of gene and protein function and interactions in the context
of living organisms – what do genes really do?
What genes do and How they do it
• Only 15-20% of human and mice genes have been knocked out using
conventional gene targeting approaches
• Remainder has essentially unknown physiological functions
Large Scale Approaches to Understanding Gene Function
• Transcriptional profiling
o Microarrays, RNAseq
• Proteomics
o Mass spectroscopy, gel electrophoresis analysis, 2 hybrid analysis,
ChlP/ChIPseq
• Functional genetics
o Gene knockout, overexpression, relplacement and mutation
Two Genetic Approaches to Determine what Genes Do
1. Reverse genetics
• Begin with cloned gene and used
knowledge to introduce mutations
back into genome in order to
investigate gene function
• For the poor / For the wealthy –
IKMP
• 1) Knocking out genes
o Manipulating the genetic makeup of an organism to either
constitutively or conditionally remove a gene
o The complete inactivation of a particular gene in an organism =
null mutation
o Take gene and knock it out, moving from gene to phenotype
o Include selective marker in target gene of embryonic stem (ES)
cell
▪ ES cell + homologous recombination → targeted gene
find more resources at oneclass.com
find more resources at oneclass.com
• 2) Over-expressing genes
o Driving aberrant gene expression generally in a tissue of
interest
o Take promoter of gene → insert into egg → expresses gene
• 3) Subtle manipulations of gene
o Altering specific amino acids, removing parts of gene,
removing parts of promoter
o Used to alter expression pattern of genes within embryo
• Problems with reverse genetic approaches
o 1) Timeframes involved
▪ Takes 6 weeks to electroporate ES cells/select colonies
→ 21 weeks to identify recombinants, inject into
blastocysts, mice born, breed from chimeras → takes 40
weeks to identify knockout mice, intercross mice,
establish lines (overall a year)
o 2) Very Expensive: high throughput reverse genetics (for
wealthy)
▪ Aim: generating knockouts ES cell for all mouse genes
▪ International knockout mouse project (IKMP) – 3
parallel initiatives
▪ Cost $50,000
▪ $10,000 – 15,000 to make transgenic mouse
▪ $40,000 – 70,000 to make gene out
• EUCOMM Approach
o 3 exons: used homologous recombination to replace middle
part of gene
o Splice acceptor, LacZ, Neor → stop codon
o Inactivate gene function
o Frt and LoxP sites
▪ DNA sequences that are recognised by two different
DNA recombinases (Flp and Cre)
▪ Recombinases will remove or duplicate the DNA in
between the DNA sequences – depending on orientation
of DNA sequence
o Why include LacZ marker:
▪ Useful: allows you to visualise where a gene is
expressed
▪ Can permit measurement of transcriptional activity
from a particular target locus
▪ Can image developing structures and organs if you
trapped gene is expressed
o Conditional allele is same as wild type gene
o Expressing Cre – can control where gene is knocked out →
produces null allele
o Can express Cre with drug
find more resources at oneclass.com
find more resources at oneclass.com
