Minimal Promoters:

Minimal promoters are used when they are well characterized and proven to result in high levels of expression in the cell type indicated.  Expression cassettes are cloned into the promoter plasmid using standard cloning techniques.  Following pronuclear injection of the minimal promoter transgene into fertilized oocytes, founder animals should express the gene product in an expression pattern similar to the gene whose promoter is controlling it.

minimal promoter	size	reference
αMHC	6 kb	http://www.ncbi.nlm.nih.gov/pubmed/2026617 pJG/ALPHA MHC was a gift from Jeffrey Robbins (Addgene plasmid # 55594)

 

BAC Recombineering:

Bacterial artificial chromosomes (BACs) are cloning vectors (7-12 kb) containing 150-250 kb of genomic DNA.  Because of the large size of insert, most BACs contain genes in their entirety including any cis-regulatory elements. By using recombineering methodologies, we are able to insert our expression cassette under control of the promoter of a particular gene.  Following pronuclear injection of the BAC transgene into fertilized oocytes, founder animals should express the gene product in an expression pattern similar to the gene whose promoter is controlling it.

Gene Promoter	BAC used size (bp)	BAC link	reference
acta2	RP23-370F21 175,077 bp	http://www.ncbi.nlm.nih.gov/clone/718411/	http://www.ncbi.nlm.nih.gov/pubmed/25414670
Aldh1L1	RP23-7M9     227,795 bp	https://www.ncbi.nlm.nih.gov/clone/602084/	https://www.ncbi.nlm.nih.gov/pubmed/18171944
cdh5	RP23-453P1 200,292 bp	http://www.ncbi.nlm.nih.gov/clone/744205/
CGRP	RP23-181A2 185,669 bp	http://www.ncbi.nlm.nih.gov/clone/656740/	http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=770
Chat	RP23-268L19 171,887 bp	http://www.ncbi.nlm.nih.gov/clone/685186/	http://www.ncbi.nlm.nih.gov/pubmed/16940431
Cx40	RP24-255O4 166,798 bp	https://www.ncbi.nlm.nih.gov/clone/812542/
dbh	RP23-354N13 188,522 bp	http://www.ncbi.nlm.nih.gov/clone/568904/	http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=619
FoxJ1	RP23-166G11 194,452 bp	https://www.ncbi.nlm.nih.gov/clone/651794/
gfap	RP24-331K9  201,171 bp	http://www.ncbi.nlm.nih.gov/clone/834100/	http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=507
HCN4	RP23-281H22 236,697 bp	http://www.ncbi.nlm.nih.gov/clone/689632/	http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=618
Lck	RP24-159E19 149,944	http://www.ncbi.nlm.nih.gov/clone/783435/
Ly6G	RP24-268F10 186,304 bp	https://www.ncbi.nlm.nih.gov/clone/870760/
PDGFRα	RP23-55P22 244,503	http://www.ncbi.nlm.nih.gov/clone/616900/	http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=1833
PDPN	RP23-146H1 193,282 bp	http://www.ncbi.nlm.nih.gov/clone/645576/
POSTN	RP23-144B14 218,286	http://www.ncbi.nlm.nih.gov/clone/645026/
SP-C	Rp23-247J9 180,528 bp	http://www.ncbi.nlm.nih.gov/clone/678272/

 

Construction of BAC transgenes (as an example, the generation of acta2-optoα1AR-IRES-lacZ is given):

a)      Construction of the target vector:  A BAC containing the promoter of interest is obtained and used as a template for PCR amplification of arm 1 and arm 2.  Arms 1 and 2 are typically 500-700 bp long and are derived from the genomic sequence immediately upstream and downstream respectively of the initiation codon of the gene (in this case the initiation codon is in exon 2).  Arms 1 and 2 are cloned into the target vector flanking the expression cassette.

 

 

b)      Homologous recombination:  The insert is purified from the target vector and used to transform bacteria carrying the BAC.  Recombination enzymes are activated and homologous recombination occurs between the BAC and the target vector.

 

c)      The resulting construct is the final transgene construct where the expression cassette has replaced the initiation codon in the BAC.