|4. WORK PLAN (max 2 A4 pages, plus tables and charts)
The Project aims to produce a MAC that can be delivered and will lead to controlled gene expression stably in vivo. Tasks here are presented in some detail. The timing is indicated in chart 1 and the exchange of materials and/or personel in chart 2
1. Determining what DNA efficiently makes centromeres in human cells and in mouse cells. HC
a)1 year Assess at least 2 new centromeric sequences for de novo centromere formation in HT1080 cells.
b)1 year Assess known centromere forming sequences in mouse ES cells
c)1 year transfer known centromere forming sequences into ES cells
2. Transfer of existing novo-chromosomes into mice and analysis. HC
a) Transfer of novo-chromosomes by microcell fusion into ES cells (1 year).
b) Transfer of ES cells into chimeric mice (1 year)
c) Analysis of stability and segregation of the novo-chromosomes in mice (1year).
3. Better constructs for telomere formation - tethering. EG
a) Making constructs with multiple sites for LacI binding (1 year).
b) Making fusion proteins (1 year).
c) Testing constructs for efficiency of telomere formation (1 year).
4. What effect do telomeres have on gene expression. (EG)
a) Introduction of constructs with a reporter gene and with or without
telomere into mammalian cells (1 year).
b) Assessment of gene expression with or without the telomere (2 years).
5. A replication cassette that can be used in MAC constructs. (AF)
a) Make various casettes containing the lamin B2 origin and assess for replication at different sites of the genome.
b) Add to chromosome 21 give to Roizes
c) to Add to synthetic MACs (1)
6. What effect do telomeres and centromeres have on replication. (SR)
a) Putting the lamin B2 origin next to a telomere or not next to a telomere using EG transfection assay (1 year).
b) Assessing the effect of telomere proximity on the origin function (1 year).
c) Assessing the effect of centromere proximity on the origin function (1 year).
7. Analysis of replication origins within a region of euchromatin and near a telomere. (SR)
a) Collecting sequences to assay over a region of chromosome 21 near centromere and telomere (1 year)
b) Scanning the large region for replication origins (2 years).
8. Construction of BAC constructs with the intact CFTR and HPRT genes with and without OriP and EBNA1. CH
a)1 year convert known YAC into a BAC with OriP and EBNA1.
b)1 year assess gene expression of the CFTR and HPRT genes
9. Construction of MACs with lox site and introduction of CFTR and HPRT genes onto this. (CH)
a) Make a MAC with lox site - modification of material from (HJC)- assess function of chromosome (1 year).
b) Introduce genes by cre/lox into the novo-chromosome (1 year)
c) Analyse expression of the genes on the novo-chromosomes (1 year).
10. Modification of deleted chromosome 21 (GR)
a) Target a loxP site to 21q by homologous recombination, confirm correct integration (1 year).
b) 1 year; Clone sequences flanking alphoid (beta satellite and satellite 1) in BACs.
c) Add known functional alphoid from (HJC). Test stability
1year - introduce construct to lox modified cell , express cre and add CFTR construct
Test stability and expression
11. Cloning and functional analaysis of centromeric DNA from a deleted chromosome 21 (GR)
a) Clone sequences flanking alphoid (beta satellite and satellite 1) in BACs (1 year).
b) Ligate known functional alphoid from (HJC) and transfect into cells (1 year).
c) Test stability and expression
12. Delivery of large DNA to primary cells in vivo. (EW)
a)1 year; deliver a large ORiP EBNA1 construct with a number of different lipid to see which ones give the best frequency of delivery and intact DNA
b)1 year deliver the large oriPEBNA1 constructs to primary cells in vivo
c)1 year determine intact ness of DNA in vivo after delivery.