Seminario acerca de mutagénesis en diferentes líneas celulares

1. Juan Esteban Restrepo Ruiz
Biologia Molecular
Facultad de Medicina UPB
2018

2. Introduction.
Mutagenesis Lymphoblasts Radiation

3. Introduction.
In the experiment they put certain cell lines to grow in different media
that were irradiated. These cells were specifically lines of
lymphoblasts and E. coli.
Finally they evaluated the mutation effects of growing in different
media that was treated with processes of radiation.

4. Objective.
Evaluate the effect of high dose irradiated meat samples (HIDMS) on
simple model systems including wild type E. coli cells and human
lymphoblast cell line.

5. Materials.
1.Procurement, processing and
gamma irradiation of meat sample
● Gamma radiation (25kGy) was carried
out at dry ice (-78.5°C) condition in a
cobalt-60 Food Package Irradiator
● Irradiation at 25 kGy is the maximum
approved dose for any food commodity
by Indian Law.
Obtain the culture media .
2. Preparation of human S9 liver
mix.
● The liver was prepared homogenizing
fresh human liver and centrifuging the
homogenate at 9000 g for 20 minutes
at 4°C
● Supernatant contains microsomes
comprised of mixed function oxidases
.
Obtain the Cytochrome P450 enzymes.

6. Materials.
3.RNA polymerase beta subunit
(rpoB) based forward mutation assay
● Wild type E. coli cells were grown in
liver S9 metabolic activated fresh meat
medium.
● Mutagenicity was assayed by rpoB
RNA polymerase based forward
mutation detection assay where cells
acquire rifampicin resistant phenotype
upon mutation in this gene
Evaluate the number of mutant E. coli
cells that acquire the rifampicin
resistant phenotype
4.DPAR mutation assay
● E. coli cells transformed with
pBR322 plasmid DNA.
● The cells grew in LB culture
media in the presence of
antibiotics (ampicillin) in the
presence of known mutagen or a
test compound and compared for
frequency of the colonies
displaying resistance to the
another antibiotics (tetracycline)
on LB-agar selection plates.
Mutagenic exposure leads to significant
variation in the antibiotics resistance
frequency of cells with respect to these two
antibiotics in a culture and observed variation
indicates the mutation frequency.

7. Materials.
5.Sequencing of pBR322 plasmid encoded
tcR gene PCR amplified from E. coli
transformed cells grown on HDIMS.
● E. coli transformants were grown
exclusively on HDIMS broth, and
subsequently subcultured till 1500
generations in the same medium
supplemented with ampicillin.
Obtain the plasmid sequencing with
PCR from the E. coli cells that grown
on the meat irradiated samples.
6. RAPD analysis of E. coli genome from
1500 generations HDIMS grown cells.
● The DNA was isolated from E. coli
using phenol-chloroform extraction.
● The DNA was subjected to RAPD
analysis using random primers.
● PCR programs.
Amplify random regions of the genome.

8. Materials.
7.Human lymphoblast mutation
(HLM) assay.
● Human Lymphoblast TK6 line.
● Analysis of mutations at two different
loci (tk+/- and hprt+).
● Ethyl methanesulfonate and 5-
azacytidine were used as mutagens
(positive controls).
This process was made to obtain the
culture media were the different cell
lines will grew up.
Obtain mutant lymphoblasts cells

9. Results. 1.Mutation frequency in E. coli MG1655 (ATCC 700926) cells.

10. Results.
2.Differential loss of Antibiotic Resistance (DPAR) assay in pBR322 transformed E.
coli MG1655 (ATCC 700926) cells and maintained up to 1500 generations
on HDIMS broth medium.

11. Results. 3.RAPD analysis of E. coli MG1655 (ATCC 700926) genome with primers

12. Results. 4.Assessment of mutagenicity in human lymphoblast cell line TK6 (ATCC CRL-8015).
tk -/+
hprt-/+
Different
irradiation

13. Discussion.
Author Idea Yes or No
Kanatt et al. 2010 Irradiation of meat samples is an
important technique to ensure its safety
and also extended shelf life.
Al-Amer et al. 2016 Among the meat samples studied,
chicken and mutton contains about six
time more fats as compared to fishes
and prawns
Saxena et al. 2016 In case of positive controls where TK6
cells were exposed to 5-AZ or EMS,
cellular aggregation was observed in
both the samples, indicating
mutagenesis
Poling et al. 1995 Three generations of
albino rats were fed electron beam
sterilized raw ground beef where no
evidence of toxicity or decrease in
nutritional value of the raw meat due to
irradiation treatment was observed

14. Conclusion.
● None of the assays indicated any mutagenesis due to HDIMS.
● The findings thus affiermed the genotoxic safety of HDIMS .
● This technology is known to have excellent potential to ensure postharvest food preservation.
➢ Food irradiation program always remained underutilized since inception. Misconceived
apprehension pertaining to the safety of irradiated food for human consumption seems to be
one of the underlying causes.
➢ Food irradiation has positioned itself as one of the best mechanisms to protect consumers from
pathogens.