Manipulation of gene expression

Question 1

Up-regulation (overexpression) techniques (2) and problems

Answer 1

Transient overexpression: short term, faster, mammalian vector, constitutive promoter produces at higher levels (not normal) selectable marker, tag protein (motif)

Stable overexpression: permanent (transfection or transduction), slower, control of the promoter (constant, induced by drug, only on specific tissue), virus inserted into 3 pieces (cDNA, envelope vector, packaging vectors)

Problems: random insertion of transgene can disrupt gene - solution multiple lines show same phenotype

Question 2

transfection vs transduction

Answer 2

Transfection - other tha viral
Transduction - viral

Question 3

Down regulation techniques

Answer 3

RNAi: reduces expression not true knock out - post transcriptional silencing (mRNA degradation - prevents translation)
1- transduction of shRNA in vector
2- process of pre-shRNA by Drosha (RNAse II)
3- transport to cytoplasm by Exportin 5
4- transformation of shRNA in siRNA by Dicer (RNase II)
or IF STARTS FROM HERE introduction of siRNA directly
5- siRNA guides Slicer (RISC) to cleave complementary mRNAs
+: cheap, transient or stable, siRNA available, targeting specific splice variants
-: off target

CRISPR-Cas9: gene knockout (Cas9+ 20nt crRNA+ tracrRNA or gRNA)
1- capture viral DNA and insert into RNA
2- crRNA protospacer sequences will recognize it
3- Cas9 cleaves DSB

Question 4

DSB repair pathways and gene knock out/knock in

Answer 4

Non homologous end joining (NHEJ) - indels with variable lenghts, no control- good for knock outs because it causes frameshits inducing premature STOP codons (PSC) - non sense mediated decay

Homologous directed repair (HDR): precise repair - good for knock in because is specific but has lower efficiency

Question 5

CRISPRi (2)

Answer 5

permanent or transient, modified version of Cas9 (dCas9) - search but don´t cleave

activation - gRNA targets promoter of gene of interest, activator domains or transcription factors recruit RNA polymerase

inihibition - dCas9 represses gene expression (better when fused to a repressor)

Question 6

Choice of method

Answer 6

short/long term to generate effect
a few days - enzyme activity (siRNA)
a few weeks - telemore length (CRISPR)

Question 7

Choice of cells (3) pros and cons

Answer 7

Primary
+: relevant (different phenotypes), available
-: from tissue, difficult and expensive, specialised media, limited lifespan

Immortalised
+: easy to grow,available, immortal
-: not normal, Chr abnormalities

STEM
+: multiple cell types, iPSCs available and can be generated from specific patient
-: difficult to grwo, specialised media, expensive, differentiation hard and not all cell types, not identical to primary, ethics