Gene technology MOB20306
case basics
restriction site must not be located in the gene but outside
DNA molecules are negatively charged and migrate towards positive electrode during gel
electrophoresis, separation occurs according to size
rRNAs are by far most abundant RNA species in cells
western blot (p)
- specific proteins are detected with labeled antibody
southern blot (d)
- DNA fragments are separated according to size and blotted with a probe
- only fragment to which the probe hybridizes is seen on the blot
- probe can only bind to single stranded DNA
northern blot (r)
- RNA molecules are separated according to size; smaller RNAs are located at bottom
- probe can be used to visualize specific RNA molecule in an RNA gel
PCR
- using one primer only: only one strand of original template gets amplified, new
strands don’t get amplified as they are identical and not complementary to the primer
- taq-polymerase: stable at high temperatures but no proofreading activity
- AA sequence is known > use degenerate primers (mixture of primers containing
different possible nucleotide sequences (for same AA)
to analyse mRNA it must be converted into cDNA (complementary DNA) with reverse
transcriptase and oligo(dT) primer (binds to poly(A) tails of mRNA molecules)
for promoter region, genomic DNA (not cDNA) needs to be obtained
cDNA is genomic DNA without all necessary noncoding regions (introns)
,UTR: untranslated region
first step of DNA cloning is usually the assembly of recombinant DNA construct in a vector
case the brain
creating transgenic mouse that overexpresses a certain gene
overexpression is acquired by inserting an extra copy of the endogenous gene into genome
endogenous: originating from within an organism
if you don’t want to change the endogenous gene, you don’t have to select for homologous
recombination
if you need to select for homologous recombination you have to work with embryonic stem
cells (oocyte most likely will not survive a procedure where it has to grow in selective
medium for a while)
genotype is determined with DNA and therefore with southern blot analysis
expression levels are determined with amount of protein and therefore with western blot
construct injected into fertilized oocytes > present in each cell of mouse > non-chimeric mice
transgenic embryonic stem cells injected into blastocoel > chimeric mice
determining expression of gene
- western blot to study protein levels
- northern blot to determine RNA levels
- RT-PCR to determine RNA levels
direct injection into oocyte > chimeric mice don’t form
select mice with only one insertion of transgene in genome because then the
offspring is genetically identical
genotype must be known before crossing mice for efficiency
loxP system elements are used to make conditional knockouts (not complete knockout mice)
signal peptide can be used to direct the protein to abnormal subcellular location
tissue specific promoter can be used to create different expression pattern for transgene
viral vector to deliver DNA construct into cell
,case asthma
make mouse model for asthma by targeted disruption of T-bet gene
when the construct is injected into the oocyte, it isn’t possible to select for homologous
recombination anymore
injecting ES cells into blastocyst > chimeric mice
, heterologous recombination = random integration/insertion
if there is a randomly integrated construct after selection for homologous recombination then
it probably took place in a transcriptionally inactive region of the genome (in ES cells) >
negative selection has no effect
PCR can’t be used to determine how many copies of construct are randomly integrated
knockout mice don’t contain the wildtype gene anymore
case recombinant DNA and fusion proteins
MCS: multiple cloning site; artificially constructed region in a vector molecule which contains
a number of closely spaced recognition sequences for restriction endonucleases that aren’t
present in other regions of the vector
sticky end enzymes create overhanging sequences (can only hybridize to complementary
overhanging sequences) > more stable product
blunt end enzymes leave blunt ends (can only ligate to other blunt ends)
case basics
restriction site must not be located in the gene but outside
DNA molecules are negatively charged and migrate towards positive electrode during gel
electrophoresis, separation occurs according to size
rRNAs are by far most abundant RNA species in cells
western blot (p)
- specific proteins are detected with labeled antibody
southern blot (d)
- DNA fragments are separated according to size and blotted with a probe
- only fragment to which the probe hybridizes is seen on the blot
- probe can only bind to single stranded DNA
northern blot (r)
- RNA molecules are separated according to size; smaller RNAs are located at bottom
- probe can be used to visualize specific RNA molecule in an RNA gel
PCR
- using one primer only: only one strand of original template gets amplified, new
strands don’t get amplified as they are identical and not complementary to the primer
- taq-polymerase: stable at high temperatures but no proofreading activity
- AA sequence is known > use degenerate primers (mixture of primers containing
different possible nucleotide sequences (for same AA)
to analyse mRNA it must be converted into cDNA (complementary DNA) with reverse
transcriptase and oligo(dT) primer (binds to poly(A) tails of mRNA molecules)
for promoter region, genomic DNA (not cDNA) needs to be obtained
cDNA is genomic DNA without all necessary noncoding regions (introns)
,UTR: untranslated region
first step of DNA cloning is usually the assembly of recombinant DNA construct in a vector
case the brain
creating transgenic mouse that overexpresses a certain gene
overexpression is acquired by inserting an extra copy of the endogenous gene into genome
endogenous: originating from within an organism
if you don’t want to change the endogenous gene, you don’t have to select for homologous
recombination
if you need to select for homologous recombination you have to work with embryonic stem
cells (oocyte most likely will not survive a procedure where it has to grow in selective
medium for a while)
genotype is determined with DNA and therefore with southern blot analysis
expression levels are determined with amount of protein and therefore with western blot
construct injected into fertilized oocytes > present in each cell of mouse > non-chimeric mice
transgenic embryonic stem cells injected into blastocoel > chimeric mice
determining expression of gene
- western blot to study protein levels
- northern blot to determine RNA levels
- RT-PCR to determine RNA levels
direct injection into oocyte > chimeric mice don’t form
select mice with only one insertion of transgene in genome because then the
offspring is genetically identical
genotype must be known before crossing mice for efficiency
loxP system elements are used to make conditional knockouts (not complete knockout mice)
signal peptide can be used to direct the protein to abnormal subcellular location
tissue specific promoter can be used to create different expression pattern for transgene
viral vector to deliver DNA construct into cell
,case asthma
make mouse model for asthma by targeted disruption of T-bet gene
when the construct is injected into the oocyte, it isn’t possible to select for homologous
recombination anymore
injecting ES cells into blastocyst > chimeric mice
, heterologous recombination = random integration/insertion
if there is a randomly integrated construct after selection for homologous recombination then
it probably took place in a transcriptionally inactive region of the genome (in ES cells) >
negative selection has no effect
PCR can’t be used to determine how many copies of construct are randomly integrated
knockout mice don’t contain the wildtype gene anymore
case recombinant DNA and fusion proteins
MCS: multiple cloning site; artificially constructed region in a vector molecule which contains
a number of closely spaced recognition sequences for restriction endonucleases that aren’t
present in other regions of the vector
sticky end enzymes create overhanging sequences (can only hybridize to complementary
overhanging sequences) > more stable product
blunt end enzymes leave blunt ends (can only ligate to other blunt ends)
