last  page PLNT3140 Introductory Cytogenetics
Lecture 13, part 1 of 2
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October 24, 2017

Learning checklist:

1. Be able to list several advantages of artificial chromosomes, as a genetic engineering strategy, versus incorporation of foreign DNA into host chromosomes.

2. Understand the main features of the following artificial chromosome technologies

Telomere-directed truncation
The ACE system
Maize Minichromosomes

A. Why do we need artificial chromosomes?

Traditionally, transformation of novel genes into plants and animals has employed various methods for delivering DNA into cells (eg. transfection, microinjection, Agrobacterium infection), but these methods all ultimately depend upon the DNA repair mechanisms of the target cell to insert the DNA into chromosomes at a random location, anywhere in the genome. For the purposes of genetic engineering in plants and animals, for gene therapy in humans, and for the study of gene function in transgenic organisms, artificial chromosomes, in principle, have several advantages:

B. Difficulties associated with Mammalian Artificial Chromosomes (MACs)

C. Common approaches

Mejia JE, Larin Z (2002) Advances in human artificial chromosome technology Trends in Genetics 18:313-319.

1. Creation of mini-chromosomes by deleting large sections of naturally-occuring chromosomes

Telomere-associated chromosome fragmentation (TACF), or telomere-directed truncation (TDT) makes it possible to replace most of a chromosome arm with a short synthetic vector. In two recombination steps, this method replaces two chromosome arms with synthetic vector sequences.  In the first step, a targeting vector contains the human alphoid satellite sequence (alpha) found at the centromere of the Y chromosome, and a selectible marker for neomycin resistance (neo), and a telomeric sequence (TEL). When this construct is transfected into human cells in culture, the alphoid satellite recombines with the alpha satellite, resulting in an acentric chromosome Y arm, and a shorter Y chromosome, in which the long arm has been replaced by the targeting vector. Recombinants are selected on neomycin.

The same strategy is repeated using a second targeting vector with telomere and a second selectible marker for guanine phosphoribosyl transferase (gpt). In this case, recombination of the targeting vector with random sites on the short arm of the chromosome occur. Recombinants are selected on media containing hypoxanthine, aminopterin and thymine (HAT).

This method has generated truncated chromosomes as small as 500 kb. So far, a minimum of 100 kb of alphoid DNA appears to be required to stably maintain these Y-derived minichromosomes in human cell culture.

2. Synthesis of de-novo chromosomes from small components

The above approach doesn't seem to work in human cells. Completely artificial human chromosomes have been constructed containing alphoid DNA and the neo  marker have been constructed. Neomycin resistant clones usually contain circular chromosomes with multiple tandem copies of the vector. Circular chromosomes are found even when telomeric sequences are added to the construct.

SATACs (Satellite DNA-based artificial chromosomes) - Artificial chromosomes can be generated by transfecting cells with vectors containing short satellite repeats and a selectible marker (eg. gpt). 

A - human chromosome 15 hybridized with centromere-specific α satellite DNA (red).

B - Insertion of exogenous marker/payload genes into chromosome 15 (yellow).
C - Amplification of sequences in transformant chromosome 15 creates an elongated 'sausage chromosome' with the de-novo formation of a second centromere (yellow and white arrows) resulting in a di-centric chromosome.
D- In-situ hybridization of 'sausage chromosome' with both
α satellite (red) and exogenous probes (green).
Growth on HAT medium selects for transformants.
When the vector has been inserted into centromeric microsatellite regions, these regions tend to undergo amplification of the repeat units, generating a new centromere. It is reasonable to suppose that selection itself favors cells  with large number of repeates for the gpt gene.

E - breakage of dicentric chromosomes (arrows).
G,H - Independent de-novo SATACS (arrows) with anticentromere staining (G) and in -situ hybridization, using probes described above.

Now, the original chromosome is dicentric, and breaks during mitosis. The breakpoint could be anywhere. Presumably, in some cells, telomerase adds telomeric repeats, making the new mini-chromosome stable.

At the end of the process, the final minichromosome is primarily composed of the foreign sequences.
from Hadlaczky G (2001) Satellite DNA-based artificial chromosomes for use in gene therapy.  Current Opinion in Molecular Theraputics 3:125-132.

D. The ACE System

Lindenbaum et al. (2004) A mamalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy.  Nucl. Acids Res. 32 e172.

1. Main components

The ACE system is a mamalian artificial chromosome system designed to solve many of the problems described above. It consistes of two components:

The two component system keeps all of the sequences necessary to maintain a stable chromosome on the Platform vector (which is necessarily quite large) while making it easy to manipulate the Targeting vector, which is small, and therefore easier to handle.

2. Platform vector

Platform ACE (A) is a mini chromosome containing multiple (typically 50 - 70 copies) of insertion sites for the targeting vector.

Copies of the insertion site lacking an insert will express the puromycin resistance gene driven by the promoter SV40p from the SV40 virus.

The attP sequence from E. coli bacteriophage Lambda is an insertion site recognized by Lambda integrase.
Lambda integrase can induce recombination (B) between a sequence containing the attB sequence (the targeting vector) and any attP site on the Platform vector (C). When a Targeting vector and a helper plasmid (not shown) containing the Lambda integrase gene are co-transfected into cells containing the Platform ACE chromosome, recombination between attB and attP will cause the Targeting vector to integrate into one or more attP sites on Platform ACE (D).

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3. Targeting vector

The targeting vector contains a gene for multidrug resistance, which will only be expressed when it inserts downstream from the SV40p promoter. Any gene of interest can be inserted into the ACE Targeting vector downstream from the mamalian CXp promoter. Any number of constructs can be made into Targeting vectors, each with a different gene as payload. Two examples are shown at right. In A, a Green Fluorescent Protein coding sequence is inserted downstram from the CX promoter. In B, the human erythropoietin coding sequence is used.  In both constructs, a tandem array of 6 units of the chicken β-globin LCR DNAseI hypersensitive site (HS4) are inserted. The HS4 sequence has been shown to act as a chromatin domain boundary. In each case then, the inserted gene should occupy its own unique chromatin domain. As well, the HS4 sequences presumably help to insulate the inserted sequence from the suppressive effects of the adjacent microsatellite DNA, which is heterochromatic.

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4. ACE Platform can accept many independent insertions

Because the ACE Platform chromosome contains numerous attP sites, in principle, a cell line containing ACE Platform could be transformed with many different constructs, each bearing a different gene. The expression of each gene can be tested at each step, so that in the final cell line, all inserted genes will have been shown to function.

5. Advantages of ACE vectors

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last  page PLNT3140 Introductory Cytogenetics
Lecture 13, part 1 of 2
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