Artificial chromosome

artificial chromosome vector

1. Artificial chromosome . vector P resented by : pushpendra singh gahlot M.sc botany U niversity of kota a g 18 5 x

2. Artificial Chromosome Vectors ● Artificial chromosomes are DNA molecules or fragment assembled in vitro from defined constituents, which guarantee stable maintenance of large DNA fragments with the properties of natural chromosomes. ● Artificial s e q u e n ci n g c h ro mo s o m e s are us e fu l f o r g e n o me p ro g r a m m e s , fo r f u n c t i o n a l c h a ra c t e rizat io n o f e n t ir e g e n o mi c r e g i o n s a n d fo r t h e tra n s duc t i o n o f nonhuman lar g e D N A s e g m e n t s i n t o h uma n a n d mammalian cells. ● Types: BAC, YAC, PAC, HAC

3. Bacterial artificial chromosome ● A b a c te r i a l a rtif i c i a l c h r o m o s o m e ( B A C ) i s a D NA construct, based on a functional fertility plasmid (or c l o n i n g in F -p l a s m id ) , u s e d f o r tr a n s f o r min g a n d bacteria, usually E. Coli. ● u p to The y a r e c a p a b l e o f c a rr yi n g a pp r o x ima te ly 300 kbp of inserted DNA sequence . E x: pBAC108L pBeloBAC11

4. C o mm o n g e n e C o m p o n e n t s of BAC O ri s:origin of replication from Ecoli f plasmid R ep E: encode a rep protein parA,parB,parC,: partitioning of F plasmid during cell division CM : chloramphenical resitance C os N :lambda phage cos site lacZ: β galactosidase gene T7:t7 bacterio phage driven promoter

5. Fig.: Bacterial Artificial C hrom o some p BAC1 8L

6. A p p l i c a t i o n s o f B A C ● Contribution to models of disease: Inherited disease ● genetic B A C s ar e n o w b e in g u t ili z e d i n mo d e ling diseases, often alongside transgenic mice. ● B A C s hav e b e e n u s e f u l i n t hi s f i e l d a s co mpl e x g e n e s may h a v e s e v e ra l r e g u l a to r y s e q u e n c e s u p s t r e a m o f t h e e n co d in g s e q u e n c e , inc l ud in g v ario u s p r o mo t e r sequences that will govern a gene's expression level. ● BACs have been used to study neurological diseases such as Alzheimer's disease or as in the case of aneuploidy associated with Down syndrome. There have also been instances when they have been used to study specific oncogenes associated with cancers.

7. Applications of BAC [contd.] ● Contribution to models of disease: Infectious disease ● T h e g e n o m e s o f s e v e ra l lar g e D N A v i r us e s a n d RNA viruses have been cloned as BACs. ● T h e s e co n s t ruc t s are r e f e rr e d t o a s " i n f e c t i o u s clones". ● The infectious property of these BACs has made the study of many viruses such as the herpesviruses, poxviruses and coronaviruses more accessible.

8. Applications of BAC [contd.] ● S e q u e n ci n g : B A Cs are o f t e n us e d t o s e q u e n ce t h e g e n o m e o f in genome projects, for example t h e H um a n o r g an i s ms G e n o m e Project. A short p i e ce o f t h e organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico , resulting in the genomic sequence of the organism.

9. Y e a s t artificial chromosome ● ● Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae Yeast artificial chromosome (YAC) vectors allow the cloning, within yeast cells, of fragments of foreign genomic DNA that can approach 500kbp in size . The YAC was devised and first reported in 1987 by David Burke

10. C onstruction of yac  YAC is built using an initial circular DNA  plasmid , which is typically cut into a linear DNA molecule using  restriction enzymes ;  DNA ligase  is then used to ligate a DNA sequence or gene of interest into the linearized DNA, forming a single large, circular piece of DNA .

11. A pplication of yac , YACs can be utilized to clone and assemble the entire genomes of an organism . it can be used to find genes, segments of DNA that code for a specific protein or phenotype , 

12. Limitations o f u s i n g Y A C v e c to r s  p r o n e to V e r y larg e DN A mo l e c u l e s ar e v e r y f rag i l e and breakage, leading to problem of rearrangement.     Dif f i c u l t to s e p ara t e Y A C f r o m t h e ot h e r h o s t c h r o mo s o m e s because of their similar size. Yi e l d o f D N A i s n ot h ig h whe n t h e Y A C i s i s o lat e d f r o m yeast cells.  C l o n e s t e n d t o b e u n s t ab l e , w i t h t h e i r f o r e ig n DN A i n s e r t s often being deleted.

13. P 1 derieved artificial chromose P 1 derived artificial chromosome are DNA cunstruct that are derived from dna of p1 bacteriophage they can carry large amount(about 100-300 kbs ) of other sequence for a variety of bioengineering purose. P1 was developed as a cloning vector by  Nat Sternberg  and colleagues in the 1990s.

14. Human artificial chromosome ● chromosome A human artificial micr o c hr o m o s o m e t h a t ca n act ( H AC ) i s a a s a n e w c hr o mo s o me in a population of human cells. ● That is, instead of 46 chromosomes, the cell could have 4 7 w i t h t h e 4 7 t h b e i n g v e r y s mall , ro u g h l y 6 - 10 m e g ab as e s ( M b ) i n s iz e f o r n a t u ra l c hr o m o s o m e s , a n d in s t e a d able o f 5 - 2 50 M b to carry n e w g e n e s introduced by human researchers. ● Ideally, r e s e arc h e rs c o u ld in t e g ra te d if f e r e nt g e n e s t h at perform a v arie ty of fu n c t i o n s , i n cl u d i ng d is e as e defense.

15. Construction m e t h o d s H A C ● T h e r e are cu r r e n t l y t w o acce p t e d mo d e l s f o r t h e creation of human artificial chromosome vectors. ➔ ➔

16. Construction methods HAC [ contd.] ● In engineered chromosome by a top-down approach, minichromosomes or chromosomes chromosomes generated d e r i v e d by fro m n a t u r al of chromosomes, telomere-directed rad iati o n - in d uc e d endogenous fra g m e n t a t i o n chromosome chromosome b r e aka g e , breakage, or c o n t a i n i n g an functional centromere. The chromosomes e n d o g e n o us ca n t h e n b e microcell-mediated tran s f e rr e d in t o o t h e r c e l l li n e s b y chromosome transfer (MMCT).

17. ● I n d e n o v o artificial chromosome chromosomes ca n b e by a bottom-up circular or n o v o fr o m cl o n e d ap p r o ac h , e x o g e n o u s linear, created de components, either naturally occurring c h r o m o s o m a l o r s y n t h e t ic high-order α -satellite bacterial artificial chromosome D N A a r r a y s i n t ro d uc e d o n ( B A C) , y e a s t art ific i al chromosome (YAC), or p1-derived artificial chromosome (PAC) vectors, which have a functional centromere and autonomously replicate and segregate. Construction methods HAC [ contd.]

18. A pp l i c a t i o n s o f H A C ● H A C s are u s e f u l i n e x p r e s s i o n s t u di e s a s g e n e tra n s f e r v e c t o rs , a s a t o o l f o r e l u c i d a t i n g h uma n c hr o mo s o me func t i o n , a n d a s a m e t h o d fo r ac t i v e l y a n n o t at i n g t h e human genome. ● HACs have been used to create transgenic animals for use as animal models of human disease and for production of therapeutic products. ● H A C c a n c a rr y g e n e s t o b e i n t r o d uc e d in t o t h e c e ll s in gene therapy.

19. Thank You a g 18 5 x