Creation of transgenic constructs

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
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
dbh RP23-354N13
188,522 bp
http://www.ncbi.nlm.nih.gov/clone/568904/ http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=619
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/
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.

 

construction of homologous recombination target vector

 

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.

homologous recombination between the target vector and the BAC

 

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

acta2-opto1AR-IRES-lacZ transgenic construct

acta2-opto-α1AR-IRES-lacZ transgenic construct

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