Editing Apoptosis (section)

==Pathway knock-outs==
Many [[gene knockout|knock-outs]] have been made in the apoptosis pathways to test the function of each of the proteins.  Several caspases, in addition to [[APAF1]] and [[FADD]], have been mutated to determine the new phenotype.  In order to create a tumor necrosis factor (TNF) knockout, an exon containing the nucleotides 3704–5364 was removed from the gene.<ref>{{cite journal | vauthors = Wang C, Youle RJ | title = The role of mitochondria in apoptosis | journal = Annual Review of Genetics | volume = 43 | issue = 1 | pages = 95–118 | date = 2009-12-01 | pmid = 19659442 | pmc = 4762029 | doi = 10.1146/annurev-genet-102108-134850 }}</ref>  This exon encodes a portion of the mature TNF domain, as well as the leader sequence, which is a highly conserved region necessary for proper intracellular processing.  TNF-/- mice develop normally and have no gross structural or morphological abnormalities.  However, upon immunization with SRBC (sheep red blood cells), these mice demonstrated a deficiency in the maturation of an antibody response; they were able to generate normal levels of IgM, but could not develop specific IgG levels.<ref>{{cite journal | vauthors = Marino MW, Dunn A, Grail D, Inglese M, Noguchi Y, Richards E, Jungbluth A, Wada H, Moore M, Williamson B, Basu S, Old LJ | display-authors = 6 | title = Characterization of tumor necrosis factor-deficient mice | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 94 | issue = 15 | pages = 8093–8098 | date = July 1997 | pmid = 9223320 | pmc = 21562 | doi = 10.1073/pnas.94.15.8093 | doi-access = free | bibcode = 1997PNAS...94.8093M }}</ref>  Apaf-1 is the protein that turns on caspase 9 by cleavage to begin the caspase cascade that leads to apoptosis.<ref>{{cite journal | vauthors = Bratton SB, Salvesen GS | title = Regulation of the Apaf-1-caspase-9 apoptosome | journal = Journal of Cell Science | volume = 123 | issue = Pt 19 | pages = 3209–3214 | date = October 2010 | pmid = 20844150 | pmc = 2939798 | doi = 10.1242/jcs.073643 }}</ref>  Since a -/- mutation in the APAF-1 gene is embryonic lethal, a gene trap strategy was used in order to generate an APAF-1 -/- mouse.  This assay is used to disrupt gene function by creating an intragenic gene fusion.  When an APAF-1 gene trap is introduced into cells, many morphological changes occur, such as spina bifida, the persistence of interdigital webs, and open brain.<ref name="auto">{{cite journal | vauthors = Mailhos C, Howard MK, Latchman DS | title = A common pathway mediates retinoic acid and PMA-dependent programmed cell death (apoptosis) of neuronal cells | journal = Brain Research | volume = 644 | issue = 1 | pages = 7–12 | date = April 1994 | pmid = 8032951 | doi = 10.1016/0006-8993(94)90339-5 | s2cid = 22542598 }}</ref>  In addition, after embryonic day 12.5, the brain of the embryos showed several structural changes.  APAF-1 cells are protected from apoptosis stimuli such as irradiation.  A BAX-1 knock-out mouse exhibits normal forebrain formation and a decreased programmed cell death in some neuronal populations and in the spinal cord, leading to an increase in motor neurons.<ref name="auto"/>

The caspase proteins are integral parts of the apoptosis pathway, so it follows that knock-outs made have varying damaging results.  A caspase 9 knock-out leads to a severe brain malformation {{Citation needed|date=October 2022}}.  A caspase 8 knock-out leads to cardiac failure and thus embryonic lethality {{Citation needed|date=October 2022}}.  However, with the use of cre-lox technology, a caspase 8 knock-out has been created that exhibits an increase in peripheral T cells, an impaired T cell response, and a defect in neural tube closure {{Citation needed|date=October 2022}}.  These mice were found to be resistant to apoptosis mediated by CD95, TNFR, etc. but not resistant to apoptosis caused by UV irradiation, chemotherapeutic drugs, and other stimuli.  Finally, a caspase 3 knock-out was characterized by ectopic cell masses in the brain and abnormal apoptotic features such as membrane blebbing or nuclear fragmentation {{Citation needed|date=October 2022}}.  A remarkable feature of these KO mice is that they have a very restricted phenotype: Casp3, 9, APAF-1 KO mice have deformations of neural tissue and FADD and Casp 8 KO showed defective heart development, however, in both types of KO other organs developed normally and some cell types were still sensitive to apoptotic stimuli suggesting that unknown proapoptotic pathways exist.{{citation needed|date=November 2024}}