A Guided Inquiry‐Based Animal Cell Culture Laboratory Course for Undergraduates
Authors: CARASTRO L. MICHAEL 
Published: November 2023

The revised version of this course, Tissue Culture (CHE470), includes a series of animal cell culture‐based experiments and lectures on Biochemistry & Molecular Biology (BMB) aspects of cell culture, including cellular transformation and metabolism, growth kinetics, transfection, inducible gene expression and biochemical assays. After receiving lectures on BMB aspects of cell culture and training in fundamental animal cell culture techniques, student undertake a unified series of experiments consists of (1) animal cell transfection by electroporation, (2) clonal selection, isolation and expansion, (3) induction of gene expression with tetracycline, (4) genomic DNA (gDNA) isolation from clonal cell populations and PCR screening of clonal gDNAs, (5) RNA isolation and Real Time‐PCR, (6) protein isolation for western blotting, and (7) beta‐galactosidase enzyme activity assays. In the revised version of this course, first executed in Fall 2015, students were provided with two plasmid DNA samples for transfection into CHO T‐RExTM cells. Each student received a known sample of an unaltered tetracyclin‐inducible beta‐galactosidase expression vector (pcDNA4/TO/mycHIS‐LacZ) and one of four pcDNA4/TO/mycHIS‐lacZ vectors altered by site‐directed mutagenesis. Students were not informed about the nature of the mutation in the mutant vector provided to them. Mutations in the four altered vectors include single nucleotide mutations (SNP) that inactivate the LacZ ATG start codon (ATG‐>TTG), or one of two missense mutations in the beta‐galactosidase active site (E461K and Y503C), or a nonsense mutation that produces a truncated beta‐galactosidase protein (Y503X). After separately transfecting their two vectors into CHO T‐RExTM cells, student used antibiotic selection and isolated clonal cell populations that they molecular characterized. Students utilized their experimental data acquired from TaqMan RT‐PCR, PCR screening of clones and direct DNA sequencing, western blotting and beta‐galactosidase activity assays to determine the exact nature of the mutation in their pcDNA4/TO/mycHIS‐LacZ mutant vector provided. This guided experimental approach provides opportunities for inquiry‐based learning through students’ acquiring and analyses of experimental observations and data. Course grades are based upon a written BMB peer‐review journal style manuscript report of their experimental data and conclusions from their semester‐long series of experiments, three in‐class exams, an oral presentation of their data and findings, and some animal cell culture laboratory exercises. Student outcomes were measured by anonymous feedback through an online survey system using a numeric rating scale (5 = strongly agree; 1 = strongly disagree). Combined Fall 2013 and 2014 survey data from CHE470 student cohorts (n = 7) reported they “know more about this subject” (mean = 5.00, dev. = 0.000), that their “skills in this area have improved” (mean = 5.00, dev. = 0.000), their “appreciation of this subject increased” (mean = 5.00, dev. = 0.000), and that “learning objectives of the course were met” (mean = 4.86, dev. = 0.378). Survey data are consistent with students finding this course informative and beneficial.Support or Funding InformationThis project was supported through generous financial support from the University of Tampa Department of Chemistry, Biochemistry & Physics.