Transgene designEach transgene contains a:

1. promoter, 2. an intron, 3. a protein coding sequence 4. a reporter gene and5. transcriptional stop sequence.

These elements are typically assembled in a bacterial plasmid.

Sequences are usually chosen from previous transgenes with proven function.

Prokaryotic sequences removed before injection into the nucleus of a zygote.

The Promoter.

Promoter is a regulatory sequence that will determine in which cells and at what time the transgene is active. It is derived from sequences of a gene upstream from the start site of transcriptionThe promoter sequence may contain• transcriptional start site • transcription regulatory sequences &• some extraneous sequence downstream of the

transcriptional start .Important: promoter expression pattern. Temporal/spatial(e.g. the heat inducible promoter, hsp17.3B showed uniform expression pattern in all cells and tissues following a mild heat shock in contrast to the CaMV 35S, )

transcription factors that are activated or repressed by environmental stimuli and/or developmental signals. The rectangular boxes labeled A, B and C represent the cis-acting factors and the ellipses labeled A, B and C represent the transcription factors. Abbreviation: Ub, ubiquitin.

The promoter DNA is the progressively digested from it's 5' end & reporter gene activity. The difference in activity between different promoter fragment lengths indicates the presence of a regulatory element in the section of DNA which was deleted.

Spinach Promoter Analysis

A region between -300 to -200 was important for enzyme induction.DNA footprinting revealed two GATA sequence elements between -300 to -130 region

Important cis regulatory elementsComponent specificcis element Sequence Function assignedERE ATTTCAAA cis-acting regulatory element involved in

ethylene-responsive elementAuxRE TGTCTCAATAAG part of an auxin-responsive elementABRE CACGTG cis-acting element involved in the abscisic

acid responsivenessP-box CCTTTTG cis-acting regulatory element involved in

gibberellin-responsive elementTATC-box TATCCCA cis-acting element involved in gibberellin-

responsivenessPlant Tissues SpecificCAT-box GCCACT cis-acting regulatory element related to

meristem expressionSkn-1 GTCAT cis-acting regulatory element required for

endosperm expressionHD-Zip 1 CAAT(A/T)ATTG element involved in differentiation of the

palisade mesophyll cells

Important cis regulatory elements: LREs

Important cis regulatory elements: ABREs

ABA (through inhibiting PP2C) phosphorylate and activate downstream transcription factors (ABF) to initiate transcription at ABA-responsive promoter elements (ABREs).

Important cis regulatory elements: HSPs

Various stresses lead to unfolding of HSPs and dissociate them from heat shock transcription factor (HSF). The freed HSF through trimerization and hyperphosphorylation binds to the heat shock element (HSE) and upregulate hsp genes.

Synthetic promoters Promoters are needed to work : • under a desired condition, • at a desired strength and • in a specified tissue

Two ways to the synthesize promoters: • One through bioinformatical model and database

(HEARTBEAT) for promoters responsive to user-defined inputs.

• Second through biochemical method to synthesize promoter libraries.

Synthetic promoters core promoter or minimal promoter contains:

1. TATA box, 2. transcription start site or CAP site

Other elements of promoter regions include:3. INR (initiator) sequences near the transcription start site;4. enhancers; and5. upstream elements

(e.g. a range of cis-acting elements are needed to mediate local gene expression in plant (boxes W1, W2, GCC, JERE, S, Gst1, and D after pathogen attack).

Synthetic promoters with specialized functions have also been developed.

May or may not haveDetectable activity

The reporter (protein coding sequence). Transgenes must contain a valid protein coding sequence (CDS) derived from the cDNA.The CDS must contain

1. a translational start codon (ATG) 2. translational stop codon &3. a Kozak sequence (5 UTR that allows ribosomes to ′

recognize the initiator codon)upstream of the start codon.

• The ideal Kozak sequence is GCCGCCACC. • Extra linker sequences (for RE) (devoid of start codons).• 5’ and 3’ non-translated sequences from the protein coding

transcript should be avoided as much as possible, since these may contain regulatory elements controlling translation or mRNA stability.

Introns 1. Integration site: transgene expression may

be more active in one site than another (factors ?).

2. Inclusion of an intron results in a significantly greater percentage of active transgenes.

(e.g. 6/7 transgenes with an intron had detectable activity, while 2/5 identical constructs without an intron had detectable weaker expression)

transcriptional stop signalEach transgene must also contain a transcriptional stop signal to match the start signal typically included in the promoter. Eukaryotic transcriptional stop signals include • a polyA addition sequence (AAAUAA) &• hundreds of downstream nucleotides whose

function is not clearly understood. Commonly used introns are -rabbit β-globin or

-SV40 (Simian vacuolating virus 40 ) &

stop sequences are from -SV40 or -human growth hormone.

Editing the transgene

Transgene sould be1. linear 2. Free from bacterial ori and 3. Free from prokaryotic sequences.

Linker and extra sequences

Typical cloning methods will have • Restriction endonuclease sites or • Plasmid polylinker sequences

These sequence must be free of • translational start or stop sites & • unwanted functional elements like enhancers

& promoters.