| Substitution of DNA | knockin | one-for-one substitution of DNA sequence information | (Targeted) Replacement of a gene, Point mutation | Protein w one aa substitution |
예전 정리 분류
| Knock-in | transgenic | ||
|---|---|---|---|
| Method | Homologous recombination | Random integration | Random integration |
| 위치 | in Jackson 2009's FFI knock-in mice, the mutant FFI Prnp gene is located in exactly the locus where Prnp is always located in mice. | the desired gene could end up anywhere in the host genome | the desired gene could end up anywhere in the host genome |
| 장점 | So the gene will achieve biological (i.e. natural) expression patterns and levels. | The desired gene might be placed under its own (strong) promoter, leading to high levels of expression, which can be good for disease models, leading to an earlier or more robust phenotype | |
| Disadvantage | it's not as faithful a model of the human disease. Eg. Prnp in mice is normally expressed more highly in neurons than in glial cells. If a Prnp transgene were overexpressed in glial cells, that would be ectopic expression and it could lead to a less accurate disease phentoype, and we might have less confidence that therapeutics effective in that mouse model would translate to humans. | ||
Cre-LoxP recombination
- A conditional knock-out method
- 어떤 (특정한 cell에서만 있는 promoter뒤에 Cre 붙이면 그 type cell 에서만 gene deletion 등 가능히다.)
- Generation method
- 조직 그리고 발생특이적인 promoter에 cre유전자를 붙여 넣은 mouse strain과 loxP site를 넣어논 mouse strain을 교배하여 hetero를 만들면 됩니다. → hetero mouse는 조직특이적으로 cre 단백질이 발현하여 loxP 부위를 잘라냅니다.
- a single Cre recombinase recognizes two directly repeated loxP site, then the Cre excises the loxP flanked (floxed) DNA, thus creating two types of DNA with circular, excised and inactivated gene Y: gene Y gene 이 두 동강이 남
- Cre
- Cre (Cre recombinase) is one of the tyrosine site specific recombinases (T-SSRs) including flipase (Flp) and D6 specific recombinase (Dre).
- it was discovered as a 38-kDa DNA recombinase produced from cre (cyclization recombinase) gene of bacteriophage P1 [3-6].
- loxP
- It recognizes the specific DNA fragment sequences called loxP (locus of x-over, P1) site and mediates site-specific deletion of DNA sequences between two loxP sites [7,8].
- loxP site is a 34 bp sequences consisting of a two 13 bp inverted and palindromic repeats and 8 bp core sequences (Figure 1A).
- Floxing
- 특정 gene의 exon 의 양 옆에 loxP를 붙이는 행위 (=flanked by LoxP),
- Recombination between LoxP sites is catalysed by Cre recombinase.
- Floxing a gene allows it to be deleted (knocked out), translocated or inverted
- Examples
- (Panicker 2022) Parkinfl/fl Exon 7 floxed parkin mice
- Resources
- (Kim et al. 2018, PMID 30671100)
- Inducible Cre-loxP system
- Temporally inducible Exogenous inducer
- doxycycline
- Temporally inducible Exogenous inducer
- Examples
- Gba D409V-loxP
- RD-DDU
- Gba D409V-loxP
TET EXPRESSION SYSTEM
- In the bacteria, there is a gene called ‘Tet gene’ that produces a protein and makes them resistant to tetracyclin.
- Tet-off expression system
- a tetracycline-controlled transactivator protein (tTA) (composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the strong transactivating domain of VP16 from Herpes simplex virus,) 가 target gene의 expression 을 조절함.
- Tetracyclin 이 없으면, tTA가 TRE에 결합을 못함. → Gene expression remains inactive..
- Tet-on expression system
- 반대로 작용함.
(A) Present: GWAS / Future: Trans-OWAS
Visible labels and caption transcribed from the figure:
- Present: GWAS — Patients, Non-patients → Genetic information → Genome → Phenome (Phenotype A)
- Future: Trans-OWAS — Patients, Non-patients → Genetic and environmental information → Genome / Epigenome / Transcriptome / Proteome / Metabolome (stacked as Genetic info. and Environmental info.) → Phenome (Phenotype A, Phenotype B, …, Phenotype Z)
| Conventional molecular biology | Single omics | Trans-omics | Layer | Measurement | |
|---|---|---|---|---|---|
| 1 | (schematic — sparse nodes on a single plane) | (schematic — denser nodes per plane) | (schematic — multi-plane network with cross-layer arrows) | Genome | NGS |
| 2 | Transcriptome | RNA-seq (NGS) Microarray | |||
| 3 | Proteome | Mass spectrometry | |||
| 4 | Metabolome | Mass spectrometry NMR |
Trends in Biotechnology
Transcription / mRNA / Structure
The structure of a typical human protein coding mRNA including the untranslated regions (UTRs)
Bar diagram (left → right): 5’ · 5’ UTR · Start · Coding sequence (CDS) · Stop · 3’ UTR · Poly-A tail · 3’
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Knock-in/transgenic table, Method row, transgenic column | Random integration | The transgenic Method cell is partly merged with the Knock-in Random integration sub-column header in the source layout; the literal label is Random integration but the column merge structure between Knock-in’s right sub-column and the transgenic column is ambiguous from the photo alone. |
| Cre-LoxP Examples bullet | Parkin<sub>fl/fl</sub> Exon 7 floxed parkin mice | The subscript token after Parkin reads fl/fl in the photo but is small and the OCR rendered it as tx/tx and wx/x; subscript not fully crisp. |
| Cre-LoxP Generation method bullet | gene Y: gene Y gene 이 두 동강이 남 | The repeated gene Y plus trailing Korean is visible but the punctuation/spacing between the two gene Y tokens is hard to verify; possibly a single label that the OCR duplicated, but kept as transcribed. |