Embryonal Stem Cells, Zinc Finger Nucleases, CRISPR & Cloning

Question 1

Why manipulate embryonal stem (ES) cells? Where are they from…

Answer 1

Derived from the primary ectoderm….

ES cells are pluripotent and can go through meiosis - thus ES cells can give rise to germline and pure transgenic mice

Question 2

Method for manipulating embryonal stem cells?

2 transgenic animals created…

Answer 2

STEP 1,

1) mate stud male and super-ovulated female
2) isolate blastocyst from uterus
3) culture blastocyst in feeder cells ( maintain pluropotency and favour growth)
4) expanded inner cell can be divided into subcultures of ES cells

5) Each ES cell subculture can be modified through vectors containing:
- gene of interest
- neomycin (neor) selectable marker, select for growth using G418

6) Southern blotting or PCR to identify cells with correct modification

STEP 2,

1) mate albino male and super-ovulated female
2) isolate blastocyst
3) transfect blastocyst with modified ES cells
4) implant chimeric blastocyst into pseudopregnant mouse
5) mate chimeric offspring with albino sibling to obtain pure transgenic line

Question 3

ES cells manipulation in creating knock-outs… how?

Answer 3

Homologous Recombination is targeted: it’s host and transgene specific, so…
Can be used to create knockout mice through
- positive-negative selection

1) Create knockout vector…
Consists of:
- neomycin selectable marker domain - if cell contains this, it won’t die in presence of G418
- Thymidine kinase… is a herpes virus - Cells that still contain this will die in ganciclovir

2) Changes introduced in only 1 chromosome
3) mate chimeric mouse with wild type to obtain homologous

Question 4

Why use zinc finger nucleases… structure, methods and steps?

Answer 4

Modified ES cells don’t work as knockouts for all species e.g rats

• Works as a dimer
• Cleaves double stranded DNA
• Consists of ‘zinc finger array’ to recognise NT sequences

1) targeted disruption
Non-homologous end joining…
-Through insertion or deletion of base pairs
- needs screening to see what you obtained as it could induce early stop codons or frame shift

2) targeted deletion
Introduce 2 zinc-finger system to flank gene of interest, results in knockout

3) gene modification
- Double stranded break favours intrinsic homologous repair
- Thus, introduce homologous donor DNA to replace what you want e.g change sequence

4) targeted insertions
Using homologous donor DNA with new sequences in the middle

Implant in pseudopregnant rat
Screen to find wanted offsprings

Question 5

What are the steps involved in CRISPR?

Incl. structural information

Answer 5

CRISPR composed of:

• Cas locus
• CRISPR array

1) Acquisition
- Viral DNA fragmented
- Fragments inserted in CRISPR array

2) Expression
- crRNA are created palindromic to viral DNA

3) Interference
- In subsequent infection… crRNA will recognise viral DNA, Cas locus site will cut viral DNA
- Immunity

Question 6

What are the types of nuclear transfer?… notable example and method

Answer 6

1) multipotent nuclear transfer:
Replacing nucleus in unfertilised egg with nucleus of ‘inner cell mass’ (normal development)

2) somatic nuclear transfer:
replacing nucleus of unfertilised egg with nucleus of adult somatic cells

Dolly, fist successful mammalian clone using nuclear transfer

• done with somatic nuclear transfer
  1) egg isolated from black faced ewe: haploid nucleus removed
  2) diploid somatic cell derived…
• induce quiescence by growth on low serum (synchrony between nucleus and egg cytoplasm), then isolate nucleus
  3) egg and nucleus fused by electroporation
  4) cultured for 6 days before implanted in surrogate mom

Question 7

Types of cloning in humans?

Answer 7

1) non-reproductive cloning:
Therapeutic uses.
- patient nucleus is fused with enucleated oocyte
- can be differentiated based on patients needs

2) reproductive cloning:
Obtaining a whole human

Question 8

Issues regarding clone health?

Answer 8

Two aspects that reduce health:

1) cells in culture likely to acquire mutation fast
2) Tetromere length

Tetromere:
repeated sequences at the end of chromosomes
- When long, Tetromere can bend and bind to certain chromosome sequences - repressing gene expression
- When shortened, Tetromere can’t reach - expression

Dolly had short Tetromeres, sheep don’t have mechanisms to elongate tetromeres. Thus, born old?

Question 9

What does manipulation of male germ line consist of?

Answer 9

Spermatogonia - the only self renaming adult cells able to be pass to subsequent generations

Easy
Method 1: mix sperm and DNA, then do in vitro fertilisation…. sperm picks up DNA and incorporates with haploid genome (works in pigs)