Week 3

Question 1

Epigenetics

Answer 1

Study of GE/phenotype changes not caused by DNA sequence changes
> Mendelian: reciprocal cross @ autosomal locus –> same phenotype despite parent allele origin/sex

Question 2

Genomic Imprinting

Answer 2

1. Mom influence first: heritable and reversible

2. 50-200 gene loci; fetal growth; cell proliferation

Question 3

DNA methylation

Answer 3

Cytosine at CpG island; DNA, S-adenosylmethionine and DNA methyltransferase needed
1. De Novo: unmarked with DNMT3A and T3B
2. Maintenance: marked by methylation –> replication –> dsDNA hemimethylated twice –> DNMT1 and VHRF1 methylate both
> RESULTS: H3 deacetylation and methylation at lysine 9 –> binding of heterochromatin protein (HP1) and chromatin condensation

Question 4

Mech of DNA methylation

Answer 4

Actin chromatin –> DNA methyltransferase –> MECP2 and sin3/Histone deacetylase recognizes methy. cytosine –> lysine methyltransferase –> HP1 –> condensed chromatin

Question 5

Case Studies

Answer 5

1. A1 methylated/silenced –> A2 expressed
2. B1 silenced without enhancer binding
   - -> ICR + enhancer –> A1 expressed
3. ICR methylated + no binding with enhancer –> enhancer binds B2 to express; A2 silenced

Question 6

Hemizygoity

Answer 6

1. Gene 1 (M exp) + Gene 2 (P exp) –> Fetal/adult somatic
2. Germline phase (Methyl marker erased)
3. Germline establishment (sperm and egg) is when methyl marker returns
4. zygotic Fertfilization (Returns to 1)

Question 7

Prader-Willis (1/12500, death by 30)

Answer 7

1. M silenced, mutated P
2. Reduced muscle tone, milk suckling, fertility and motor skills; increased weight gain
3. Decrease in SNORD116 (Smaller nucleolar organizing RNA gene) 29 copies; SNORD115 –> autism
4. Types
   > 3-5Mb deleted in P 15q11-13
   > Maternal disomy at Ch15
   > small 15q11-13 deletion –> silencing

Question 8

Angelman (1/16000)

Answer 8

1. P silenced, mutated M
2. Developmental delay, outburst of laughter, low mental capacity, happy
3. Types:
   > deleted UBE3A (ubiquitin protein ligase E3A) Ch15
   > paternal disomy at Ch15
   > UBE3A silencing (simple mutation/P-mutation)

Question 9

X-Inactivation (All mammals, female selective)

Answer 9

1. Late blastocyst of Xi –> calico
2. XIST at xq13.2
   > 17 kb non-coding, spliced and Poly-A RNA
   > Hypermethylated Xi CpG
   > Hypomethylated histones tight (lots of macroH2A1.2 histones) –> Xi DNA + Barr Bodies –> most genes on Xi silenced and replicated
3. Results:
   > 1:1 of two cell types in females
   > Skewing (75-95%) inactivation of same chromosome –> recurrent spontaneous abortion, anemia, extreme pigment defect, deafness

Question 10

Certain X- genes are always expressed

Answer 10

1. Long Interspersed Nuclear element (LINE1): XIST-specific recognition site (in rest of q arm)
2. Genes that escaped XIST binding in Xp and/or Y (no dosage compensation)

Question 11

Environmental Influence

Answer 11

1. Twin genome: same DNA, different methylation
   > Age-related: depends on time spent apart
2. Rats exposed to stress –> pathologically aggressive adults; Maternal care

Question 12

Cell Lines

Answer 12

1. Primary: not immortalized
2. Established: immortalized; media + space in Nitrogen
   > Transfection: cells take up DNA; plasmid DNA for GE –(endocytosis)–> host cytoplasm degradation
   > Transduction: virus (target gene + promoter) in cell; microinjection of DNA (tedious)

Question 13

GFP/Luciferase

Answer 13

1. Tell tissue-specific promoter activity (cDNA)
2. determines GE during development
3. Fuses to protein –> chimeric –> study cell localization

Question 14

In Vitro Organoid Culture

Answer 14

1. Whole: bud-branching after dissection
2. Miniature Tissue: mammary gland + lymph node + 3D
3. Stem cell and 4. Primary cells + culture insert

Question 15

Transgenic Mice (KO)
*All DNA in GL cells (genetically modified)

Answer 15

Homologous Recombination
1-2-3-4 + NEO-DTA –> 1-2-NEO-4 + 3 (intron)
> DNA insert from HR into ICM of blastocyst from ES cell line –> select –> reimplant into foster mother
> Egg –> rotational cleavage –> 4-cell –> morula –> tight junction in compacted morula –> grow in mature blastocyst with 64-200 cell stage and ICM –> cross with heterozygote to identify AOI

Question 16

Transgenic Mice (KI)

Answer 16

1. UBE3A-YFP for Drug Screen:
   > Topoisomerase reactivates UBE3A-YFP –> Pat-gene not silenced to make up for mutated Mat gene
2. Expressed exchanged gene at WT locus (HDR)

Question 17

DNA Construct

Answer 17

1. Strong DNA promoter activates cDNA transcription
2. Microinjected DNA in fertilized eggs (pronuclear stage with nicked cDNA and DNA clone integration) –> transgene insert, cultured and implant in embryo
3. Detect overexpression, find key regulatory element, and express particular protein

Question 18

Immunodeficient Mice (No transplant rejection)

Answer 18

1. NSG mice: no B cells, mature T cells and NKT cells
2. PDX (patient-derived xenograft): tumor tissue grown in ID mice to mimic environment
3. Humanized-xenograft modes: co-engraft tumor and peripheral BM/blood cells into NOD/SCID mice to reconstitute murine IS cells

Question 19

CRISPR-Cas9 (Sequence-specific gene control)

Answer 19

1. Spacer matches foreign sequence
2. Cas9: DNA endonuclease guided by 2 RNAs
   > protospacer: short foreign sequence fragment
   > PAM: protospacer adjacent motif
   > Now combines tracrRNA and crRNA