FINAL MOLECULAR BIO

Mutagenesis - Dr Chau De Ming 

1. How homolog study works for a newly discovered gene 

Compare unknown gene sequence with known gene sequence in other organism 

Use gene seq database 

Show similarities 

Reveal protein function of new gene 

Predict potential roles in cellular processes and pathways 

2. How reporter gene works 

Track timing and location of a protein expression 

Green fluorescent protein 

Observation of GFP fluorescence when gene of interest is expressed 

Enable detection of gene and provide indication of transcriptional activity of the gene 

3. Germ line mutation:

First appear in gamete 

Passed from parents to offspring 

Found in every cell of offspring 

Somatic mutation:

First appear in somatic cells 

Not inherited/passed 

Found in specific cells where mutation first originated 

4. Point mutation:

Affects single ntd base pair 

Silent 

Missense 

Nonsense 

DNA mutation:

Heritable change in DNA sequence 

Frameshift 

Point mutation 

5. Gene mutation:

Change in ntd seq 

Negative effect 

Rare 

Acquired or inherited 

Sickle cell anemia 

Gene polymorphism: 

Single ntd change 

Harmless 

More common 

Inherited 

Gender, ABO blood group 

6. Types of mutation

Nonsense: intended protein is not produced due to a premature translation stop; the shorter polypeptide is usual degraded

Missense: a different amino acid changes the 

structure and function of the protein

Frameshift: addition/deletion of ntd → completely different protein, degrade immediately 

Chromosome duplication: additional copy of a segment → same gene copy few times → over expression 

Chromosome deletion: disappearance of gene→ gene no longer express → under/no expression 

Chromosome translocation: fusion of two gene fragments → new gene but mutated 

Chromosome inversion: reversal of a segment within a chromosome 

Chromosome ring: circular chromosome 

Chromosome aneuploidy: abnormal number of chromosome

Dr Shariza 

1. Define potency: ability to differentiate into different cell type 

2. Multipotent stem cell: ability to differentiate into limited number of types from 1 germ layer 

Ex: amniotic fluid stem cell, neural stem cell, hematopoietic, mesenchymal, cancer 

3. Pluripotent stem cell: ability to differentiate into all 3 germ layers, except placenta 

Ex: embryonic stem cell, induced pluripotent stem cell 

ESC: 

From embryo - inner cell mass/epiblast

Direct isolation from IVF embryo 

Embryonic destruction 

Ethical issues 

No reprogramming 

Autologous and allogenic 

Form teratoma 

Stable 

Efficient 

Homogenous 

Potential application: 

Human - Regenerative medicine, fundamental studies of disease model, drug screening and discovery 

Animal - Study basic fundamental and disease development process, understand mechanism behind differentiation 

Treatment - Parkinson's, diabetes z traumatic spinal cord injuries z Purkinje cell degeneration, Duchene Muscular dystrophy, heart disease, vision and hearing loss 

iPSC: 

From somatic cell

No embryonic destruction 

No ethical issues 

Reprogramming 

- genetic (TF): use viral or non viral vector to insert defined pluripotent genes 

- chemical: use small molecule or culture condition 

Patient and disease specific 

Autologous 

Form teratoma 

Less stable 

Less efficient 

Heterogeneous 

4. Adult stem cell

Ex: HPSC, mesenchymal, neural, epithelial, skin 

Properties: 

Non tumorigenic - can directly inject into body 

Stimulate paracrine effects 

Homing properties 

Immunomodulatory 

Autologous and allogenic 

ASC vs ESC - 

Heterogeneous 

Small amount - difficult to isolate and purify 

Limited differentiation capacity 

More DNA abnormalities 

Function: 

Repair damaged tissue and regenerate new 

Facilitate angiogenesis 

Anti inflammatory 

Halt destructive immune response 

Reduce size of scar tissue 

Prevent premature cell death 

5. GFP gene function 

GFP = reporter gene 

Expression of GFP = presence of neural precursor cells 

Sox 1 = marker for npc 

SOX 1 in host, egfp design in vector, X bal cut upstream & downstream arm, vector + host, SOX 1 = house, gfp = guest

6. Define

Transgenic: gene of choice artificially insert into genome 

Transgenic ESC: ESC artificially introduced in transgene - carry reporter gene 

Transgene: gene of choice 

Types: 

Random insertion occurs through non homologous recombination 

Targeted insertion occurs through homologous recombination, specific integration 

Inducible and cell specific transgene - random/specific 

Transgenesis: process of introducing transgene into a living org 

7. In vivo 

Pronuclear micro injection

Embryonic stem cell 

Somatic cell nuclear transfer 

Sperm mediated gene transfer 

Virus mediated gene transfer 

Yeast artificial chromosome 

8. In vitro 

Direct DNA transfer 

DNA mediated transfection 

Viral transduction 

9. Neural stem cell/amniotic fluid stem cell→ neural precursor cell

10. How to generate transgenic mouse with transgenic ESC 

Transgene artificially introduced in ESC 

Isolate transgenic ESC 

Inject into blastocyst 

Implant into uterus of surrogate mother 

Select chimeric organism 

Breed until transformed DNA found in germ line 

11. Application of transgenic mouse 

Models to study human disease - Alzheimer's 

Pharmaceutical 

Gene function analysis 

12. Factors to consider for transgenesis

Expression of the transgene 

Regulatory elements 

Monitoring strategy 

13. Transgenic microbes 

Facilitate crop growth 

Produce recombinant insulin 

Produce chemicals toxic to pests 

Potential use for gene therapy: cystic fibrosis 

14. How dolly is created 

Somatic cell nuclear transfer 

Mechanical removal nucleus of Scottish Blackface + Finn Dorsett nucleus → electrical pulse → fuse → blastocyst transplant into surrogate mother 

15. SCNT advantage and disadvantage 

Advantage: uniform models, preserve endangered species, patient specific treatment 

Disadvantage: low success rate, high rates of birth defects, shorten lifespan, ethical issues 

16. Molecular bio techniques 

PCR analyze gene expression of stem cell markers 

Western blot detect stem cell related protein 

Immunocytochemistry visualize cellular markers using specific antibody 

Flow cytometry quantify stem cell surface marker 

RNA sequencing profile global gene expression 

Karyotyping assess genomic stability and integrity 

17. Exosome based therapy 

Advantage: easy to manufacture & transport, stable, lyophilized, lower tumor risk, reduced immune rejection, no immuno compatibility issue, can be accurately measured, standardized dosing 

Disadvantage: limited clinical evidence, cargo variability, large scale production challenge, regulatory challenge 

Dr Zul 

20 steps produce glow in dark mouse 

1. Identify the gene of interest, select GFP gene as reporter gene 

2. Isolate GFP gene from jellyfish 

- extract gene by homogenization

- treat with mild detergent for cell lysis 

- remove unwanted component - centrifuge remove debris 

- salt + alcohol precipitation of nucleic acid 

- purify - ultracentrifugation - density gradient or column chromatography 

- detection by gel electrophoresis - confirm DNA presence 

- Quantification 260nm/280nm = 1.8 with spectrophotometer 

3. Select vector - virus/mammalian plasmid 

4. Treat with Type 2 restriction enzyme create compatible sticky end 

5. CIAP treatment prevent self ligate by remove 5' phosphate 

6. Ligation of vector and GFP with T4 DNA ligase 

7. Prepare competent host cell - Treat E coli with calcium chloride 

8. Transformation - heat shock 42°C 45 sec then recover in liquid broth 

8. Selection for transformation - fluorescent color selection - fluoresce green under UV via direct visualization 

9. Colony hybridization screen for colonies with recombinant GFP vector 

After confirm, grow the bacteria and extract GFP plasmid 

Transgene artificially introduced in ESC by electroporation 

Isolate transgenic ESC 

Inject into blastocyst 

Implant into uterus of surrogate mother 

Select chimeric organism 

Breed until transformed DNA found in germ line