Design Your Transgenic Construct

Standard transgenic constructs should contain all the 5' and 3' regulatory elements necessary for transgene expression; your gene of interest and/or marker gene; and restriction sites that allow isolation of a full length linear transgenic fragment for microinjection. Addition of sequences that potentially increase the level of transgene expression should be considered. A strategy for the detection of your transgene or its product (i.e., epitope tags on transgenic protein) should also be inherent in the construct design.

Keep in mind that the transgene should be a complete transcriptional unit containing all elements required for gene expression. Once integrated, the transgene must be distinguishable from the endogenous gene and/or it's expressed products, and the entire transgenic fragment must be excised from the vector with as little vector sequences retained as possible.

The purified linear plasmid-based transgenic microinjection fragment should range in size from 2-25Kb and contain no more than 100bp vector sequences.

BAC transgenic constructs range in size from 70-250Kb and can be injected in linearized or circular form. Integration rates are similar to smaller transgenes but fewer copies integrate. However, BAC transgenes are less susceptible to position effect variegation.

Transgene as a Complete Transcriptional Unit

Promoters/Enhancers

Tissue-specific promoters/enhancers: perform a literature search for previously identified tissue-specific promoter
Ubiquitous promoters: ie, chicken β-actin, cytomegalovirus (CMV) enhancer (CCAG or CAG promoter), histone H4 promoter

3'UTR

cDNA must have polyA tail for protein translation
Genomic sequences with strong termination & poly A signals: ie, bovine growth hormone (bGH), SV40, rabbit β-globin, 3' mouse protamine UTR/ polyA

Sequences That Can Influence Expression of Integrated Transgene

Positive effects:

Introns: increase efficiency of RNA processing and nuclear transport associated with RNA splicing
Heterologous introns: BGH, SV40 intron
Insulator sequences: overcome position effect variegation (Ref??)

Negative effects:

Vector sequences remaining in the microinjection transgenic fragment do not affect transgene integration rates but can significantly affect transgene expression; they can be recognized as foreign, thereby targeting the transgenic sequences for transcriptional inactivation
Expression of integrated transgene constructs containing less than 100 bp of vector sequence are generally not affected

Gene of Interest Sequences

Genomic DNA (exons & introns)
cDNA (lacking introns)
Anti-sense RNA

Marker Genes Track Transgene Expression

Types of Marker Genes

Lac/Z (from E. coli, encodes ?-galactosidase) or alkaline phosphatase: simple, protein produces can be visualized using histochemical or immunocytochemical methods at single cell level but not very sensitive
Luciferase (from firefly): very sensitive but must homogenize tissue to assess expression
Fluorescent proteins: very sensitive, visual, no staining required, analysis in live tissue, isolation of cells using FACS, several different fluorophores available
- EGFP (green, from jelly-fish)
- EYFP (yellow)
  - Venus
- ECFP (cyan)
- Red FP (red, from reef coral): new variants with longer wavelengths, lower background, increased brightness and photostability (Ref)
  - tdRFP (tandem dimer version of mRFP1)
  - tdTomato
  - DsRed-Express (specific mono/polyclonal antibodies that do not cross-react with EGFP are available and can be used for immunohistochemical analysis)

Expression

Expressed as fusion protein
Independent expression from IRES
Source of sequences (absence of cryptic splicing sequence)

Detection of Transgenic Gene, Transcript or Protein From Endogenous Gene

Detection of transgenic gene:

DNA genotyping of founder/offspring using:

species-specific/transgene-specific probes (such as human gene or gene element)
marker genes
mini-gene
unique restriction site (SNP) that can be used to distinguish between endogenous and transgenic gene
- Methods
- PCR assay; fast method but can be unreliable for founder genotyping
  - Design primers that detect sequences unique to transgene
  - Design transgene-specific probes that bridge gene-control element junction
- Southern Blot analysis; useful method for:
  - Founder genotyping
  - Analysis of transgene integration site(s)
  - Determining transgene copy number and orientation (head to tail array)
  - Assess homozygosity of transgenic animals

Detection of expressed gene product:

Expression of unique transgenic mRNA
Transgene may encodes truncated or shortened RNA
Not recommended for founder analysis

Detection of transgenic protein:

Expression of unique transgenic protein
Detection of expressed epitope/peptide tag (i.e., myc, FLAG, HA)
Detection of protein epitope using monoclonal antibodies (i.e., FACS, Western blot)