Artifical chromosomes

Question 1

Mammalian artificial chromosomes (MACs)

Answer 1

MACS = platform for loading an delivery of large synthetic genetic construct into mammalian cells

• Can hold large amount of DNA, almost no upper limit on exognous DNA size
• transferred between cell lines, even between species –>Large amounts of DNA transferable between cell lines
• Lack viral sequence –> reduced immunogenic responses
• orthogonal system
• control on copy number
• additional regulatory layers

Question 2

Requirement for MACs?

Answer 2

A linear MAC has Centromere and Telomere

1. Centromere:
for attachment of chromosomes to the mitotic spindle plus their segregation during mitosis
–> need of centromeric region to seperate during mitosis

2. Telomere
short repeat sequences at chromosme end. Special proteins bind these sequences forming a cap that prevents chromosome end from being recognizes as a double strand break.
–> important to keep chromosome functial over time, after cell devision.
–> avoid that chromosomes fuse

3. Origing of replication

Question 3

Generation of MACs

Answer 3

Can be linear or circular, depending on method used for generatin them

• Top-down approach
  –> Shortten existing chromosome and reengineer it. (No extra chromosome)
• Bottom-up approach
  –> build a nnew chromosome from scratch. 1 extra chromosome